Fluorescent pyrene compounds

ABSTRACT

The present invention relates to fluorescent pyrene dyes in general. The present invention provides a wide range of fluorescent dyes and kits containing the same, which are applicable for labeling a variety of biomolecules, cells and microorganisms. The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis, and/or treatment of disease conditions.

BACKGROUND OF THE INVENTION

Fluorescent dyes are widely used in biological research and medicaldiagnostics. Fluorescent dyes are superior to conventional radioactivematerials because fluorescent dyes are typically sufficiently sensitiveto be detected, less expensive and less toxic. In particular, adiversity of fluorophores with a distinguishable color range has made itmore practical to perform multiplexed assays capable of detectingmultiple biological targets in parallel. The ability to visualizemultiple targets in parallel is often required for delineating thespatial and temporal relationships amongst different biological targetsin vitro and in vivo. In addition, the generation of a wide range offluorescent dyes has opened a new avenue for conducting high-throughputand automated assays, thus dramatically reducing the unit cost perassay. Moreover, the low toxicity of fluorescent dyes provides ease ofhandling in vitro, and also renders it safer for imaging biologicalactivities in vivo.

Despite the various advantages of fluorescent dyes, there remains a needfor dyes with properties that are well suited for use in moderninstrumentation and in various applications. For example, a high quantumyield is generally desired when used in conjunction with common andcommercially available laser excitation sources, and there remains aneed for dyes which are capable of being excited.

SUMMARY OF THE INVENTION

Thus there remains a considerable need for improved compositions andmethods that would allow convenient and effective labeling of a widerange of molecules in various applications. The present inventionaddresses this need and provides additional advantages.

Accordingly, the present invention provides fluorescent compounds whichmay have any or all of the following characteristics. In one aspect,labeled biomolecules prepared using fluorescent compounds of theinvention show significantly reduced dimer formation. In other aspects,compounds and labeled biomolecules of the invention show other desirableproperties such as higher water solubility, improved fluorescencequantum yield, improved photostability, relatively simple synthesis,improved specificity of the labeled conjugates, and/or improved in vivohalf-life.

In one aspect, the invention provides a compound of Formula I:

wherein:

-   -   A is —O— or —NR₂—, where R₂ is —H or alkyl;    -   each R₁ and R₁′ is independently —H or alkyl, where each alkyl        may be the same or different and where any pair of two alkyl        groups may combine to form a cyclic ring;    -   a is an integer between 2 and 20;    -   L is a bond or a covalent linker comprising between 1 and 100        atoms; and    -   R_(x) is a a reactive group capable of forming a covalent bond        upon reacting with a reaction substrate.

In one embodiment, A is —O—. In another embodiment, A is —NH—.

In other embodiments, a is between 2 and 10. For example, a is 5. In oneembodiment, each R₁ and R₁′ is H.

In one embodiment, L is a bond. In another embodiment, L comprises awater-soluble moiety. In yet another embodiment, R_(x) is a reactivegroup capable of forming a covalent bond by reacting with an aminegroup. For example, R_(x) is an activated ester. Alternatively, R_(x)has the formula —CO-(Lg), wherein Lg is a leaving group. For instance,Rx is an N-hydroxysuccinimide ester or aminooxy.

In a further aspect, the invention provides a kit comprising: i) thecompound of the invention; ii) a buffer; iii) materials or devices forpurifying conjugation products; and iv) instructions instructing the useof the compound.

In another aspect, the invention provides a biomolecule comprising alabel derived from a structure of Formula I, wherein the at least onereactive moiety of Formula I has undergone a reaction which attaches thelabel to the biomolecule. In some embodiments, the biomolecule comprisesa polynucleotide. In some embodiments, the biomolecule comprises apolypeptide. In some embodiments, the polypeptide further comprises anantigen binding site. In some embodiments, the polypeptide is a wholeimmunoglobulin. In some embodiments, the polypeptide is a Fab fragment.

In another aspect, the invention provides an immunoglobin comprising alabel derived from a structure of Formula I, wherein the at least onereactive moiety of Formula I has undergone a reaction which attaches thelabel to the immunoglobin, wherein the immunoglobin is an antibody thatbinds specifically to an antigen on a cancer cell. In some embodiments,the antibody binds to erb2.

In another aspect, the invention provides a method of preparing alabeled biomolecule comprising reacting a compound having a structure ofFormula I and a substrate biomolecule under conditions sufficient toeffect crosslinking between the compound and the substrate biomolecule.In some embodiments, the substrate biomolecule is a polypeptide, apolynucleotide, a carbohydrate, a lipid or a combination thereof. Inother embodiments, the substrate biomolecule is a polynucleotide.

In yet another aspect, the invention provides a method for labeling acell within a population of cells whereby the cell is differentiallylabeled relative to neighboring cells within the population, the methodcomprising contacting the cell with a biomolecule labeled according tothe methods of the invention, wherein the biomolecule comprises atargeting moiety that binds to a binding partner that is indicative ofthe cell, and thereby differentially labeling the cell relative toneighboring cells within the population. In some embodiments, the methodfurther comprises the step of imaging the cell, the imaging stepcomprising: i) directing exciting wavelength to the cell; and ii)detecting emitted fluorescence from the cell. In some embodiments, thelabeling takes place in vitro. In other embodiments, the labeling takesplace in vivo.

In another aspect, the invention provides a method of labeling apolypeptide comprising: forming a complex that comprises the polypeptideand a binding agent, wherein the binding agent comprises a fluorescentlabel derived from a structure of Formula I, wherein the at least onereactive moiety of Formula I has undergone a reaction which attaches thelabel to the binding agent. In some embodiments, the binding agent is anantibody. In some embodiments, the complex comprises (a) a primaryantibody that binds to the polypeptide, and (b) the binding agent whichfunctions as a secondary antibody exhibiting binding capability to theprimary antibody. In some embodiments, the labeling occurs on a solidsubstrate. In some embodiments, the labeling occurs intracellularly. Insome embodiments, the complex yields a signal to noise ratio greaterthan about 100, wherein the signal to noise ratio is calculated by theformula: (fluorescent signal from a complex comprising the polypeptidebound by a primary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent). In other embodiments,the complex yields a signal to noise ratio greater than about 250,wherein the signal to noise ratio is calculated by the formula:(fluorescent signal from a complex comprising the polypeptide bound by aprimary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent). In yet otherembodiments, the complex yields a signal to noise ratio greater thanabout 270, wherein the signal to noise ratio is calculated by theformula: (fluorescent signal from a complex comprising the polypeptidebound by a primary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is the absorption and emission spectra of compound 2 of theinvention and Alexa fluor 405 in PBS. The slight red-shift of thespectra of compound 2 makes the dye more compatible with the opticaldetection system on instruments equipped with a 405 nm laser.

FIG. 2 is the absorption and emission spectra of compound 2 conjugatedto goat anti-mouse IgG according to the invention and Alexa fluor 405conjugated to goat anti-mouse IgG in PBS. The slight red-shift of thespectra of compound 2 makes the dye more compatible with the opticaldetection system on instruments equipped with a 405 nm laser.

FIG. 3 is a flow cytometry histogram showing the relative fluorescencelevels of Jurkat cells stained with various fluorescently labeledantibodies. The cells were first labeled with mouse anti-human CD3antibody and then stained with goat anti-mouse IgG labeled with Compound2 or Alexa Fluor 405 at an indicated degree of labeling (DOL) (darkcolumns). To measure the background fluorescence from each labeledsecondary antibody, the staining experiments were also carried out usingan isotype primary antibody to replace the CD3 antibody (gray columns).

FIG. 4 shows cellular microtubule staining using an alpha-tubulinprimary antibody and a secondary antibody labeled with compound 2 (whitefilaments). The DOL is approximately 5.4.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Dyes of the invention may be particularly useful for use withcommercially equipped excitation sources and detectors. For example, the405 nm diode laser line excitation source is common in most majorfluorescence-based life science instruments due to its reliability andlow cost. Dyes such as Cascade Blue acetyl azide, (see, e.g. U.S. Pat.No. 5,132,432) and Alexa Fluor 405 have both been used as suitable dyesfor the 405 nm laser line. Such dyes have absorption peak wavelengths of396 and 399 nm, respectively, which are not ideal. Moreover, theemission maximal wavelengths of the dyes (410 nm for Cascade Blue and425 nm for Alexa Fluor 405) are not well centered within the opticaldetection window on some of the popular instruments, such as the BDLSRII flow cytometer from Becton Dickson with a 450/±20 nm band filter.This results in a large portion of the emission peak for both CascadeBlue and Alexa Fluor 405 being outside of the detection window. Theabsorption and emission peaks of dyes of the invention are moreoptimally compatible with the existing instruments. In one aspect, theabsorption maxima of the dyes according to the invention are much closerto the 405 nm line and thus the dyes are more efficiently excited.Moreover, the emission maxima of the dyes are better aligned with thedetection window of common instruments (Table 1 and FIGS. 1 and 2). Thisred-shift makes the dyes more detectable on some of the most popularinstruments, such as the BD LSRII flow cytometer (FIG. 3).

The present invention discloses fluorescent compounds which may haveadditional desirable properties such as restricted intramolecularmobility, increased fluorescence quantum yield, decreased aggregation,increased solubility, decreased quenching and increased in vivo and invitro stability. The compounds may be used for labeling molecules andbiomolecules such as polypeptides and polynucleotides and are suitablefor use in a wide range of applications, including diagnostic andimaging systems.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

Fluorescent compounds and labeled molecules of the invention may exhibitreduced aggregation. Dye aggregation is often seen as a majorcontributing factor to fluorescence quenching. Prevention of aggregationin the present invention may be achieved without the use of an excessivenumber of negatively charged sulfonate groups. This in turn may aid inthe labeling of biomolecules such as proteins because the labeledprotein may have an isoelectric point comparable to that of thesubstrate protein, and may thereby better maintain its biologicalspecificity. The use of water soluble polymers of the invention may alsoaid in camouflaging or shielding the fluorophore to which it is linked.For example, such a water soluble polymer may be a relatively largegroup such as a polyethylene glycol moiety. This may be particularlydesirable when the fluorophore is linked to a biomolecule such as apolypeptide.

Definitions:

The compounds of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, NewYork, 1994, pages 1119 1190), and occur as racemates, racemic mixtures,and as individual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, being included in the presentinvention. In addition, the compounds disclosed herein may exist astautomers and both tautomeric forms are intended to be encompassed bythe scope of the invention, even though only one tautomeric structure isdepicted.

When any variable (e.g. R, L, (R₁)_(a), (L)_(q)) occurs more than onetime in any constituent, its definition on each occurrence isindependent at every other occurrence. Combinations of substituents andvariables are permissible only if such combinations result in stablecompounds. Lines drawn into the ring systems from substituents indicatethat the indicated bond may be attached to any of the substitutable ringcarbon atoms. If the ring system is polycyclic, it is intended that thebond be attached to any of the suitable carbon atoms on the proximalring only. Substitution of a ring by a substituent generally allows thesubstituent to be a cyclic structure fused to the ring.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.The phrase “optionally substituted with one or more substituents” shouldbe taken to be equivalent to the phrase “optionally substituted with atleast one substituent” and in such cases the preferred embodiment willhave from zero to three substituents.

As used herein, “alkyl” is intended to include both branched,straight-chain, and cyclic saturated aliphatic hydrocarbon groups. Alkylgroups specifically include methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, and so on, as well as cycloalkyls such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydronaphthalene,methylenecylohexyl, and so on. “Alkoxy” represents an alkyl groupattached through an oxygen bridge.

The term “alkenyl” refers to a non-aromatic hydrocarbon group, straight,branched or cyclic, containing at least one carbon to carbon doublebond. Alkenyl groups include, but are not limited to, ethenyl, propenyl,butenyl and cyclohexenyl. The straight, branched or cyclic portion ofthe alkenyl group may contain double bonds and may be substituted if asubstituted alkenyl group is indicated.

The term “alkynyl” refers to a hydrocarbon group, straight, branched orcyclic, containing at least one carbon to carbon triple bond. Alkynylgroups include, but are not limited to, ethynyl, propynyl and butynyl.The straight, branched or cyclic portion of the alkynyl group maycontain triple bonds and may be substituted if a substituted alkynylgroup is indicated.

As used herein, “aryl” is intended to mean any stable monocyclic orpolycyclic carbon ring of up to 7 atoms in each ring, wherein at leastone ring is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl oracenaphthyl. In cases where the aryl substituent is bicyclic and onering is non-aromatic, it is understood that attachment is via thearomatic ring.

The term “heteroaryl”, as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of 0, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,pyrrolyl, tetrahydroquinoline, xanthenyl, and coumarinyl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl” as used herein is intended tomean a 5- to 10-membered aromatic or nonaromatic heterocycle containingat least one heteroatom which is O, N or S. This definition includesbicyclic groups. “Heterocyclyl” therefore includes the above mentionedheteroaryls, as well as dihydro and tetrahydro analogs thereof. Furtherexamples of “heterocyclyl” include, but are not limited to thefollowing: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl.

The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl andheterocyclyl substituents may be unsubstituted or unsubstituted, unlessspecifically defined otherwise. For example, an alkyl group may besubstituted with one or more substituents selected from OH, oxo, halo,alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl orpiperidinyl.

The terms “halo” or “halogen” are intended to include chloro, fluoro,bromo and iodo groups.

The term “aromatic” is used in its usual sense, including unsaturationthat is essentially delocalized across multiple bonds, such as around aring.

The term “substituent” refers to an atom, radical or chemical groupwhich replaces a hydrogen in a substituted chemical group, radical,molecule, moiety or compound.

“Spiro” as used herein, refers to a cylic moiety which is attached toanother group such that one of the ring atoms of the cyclic moiety isalso an atom of said other group. A non-spiro substituent is a moietycyclic or noncylic which is directly attached to said other group viabond connection between atoms of the non-spiro moiety and said othergroup. An example of a spiro moiety is, for instance, a subsitutuentRing B on cyclohexanone Ring A.

Unless otherwise stated, the term “radical”, as applied to any moleculeor compound, is used to refer to a part, fragment or group of themolecule or compound rather than to a “free radical”. A radical may belinked to another moiety through a covalent bond.

The terms “polynucleotides”, “nucleic acids”, “nucleotides”, “probes”and “oligonucleotides” are used interchangeably. They refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.“Polynucleotide” may also be used to refer to peptide nucleic acids(PNA), locked nucleic acids (LNA), threofuranosyl nucleic acids (TNA)and other unnatural nucleic acids or nucleic acid mimics. Other base andbackbone modifications known in the art are encompassed in thisdefinition. See, e.g. De Mesmaeker et al (1997) Pure & Appl. Chem., 69,3, pp 437-440.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear, cyclic, or branched, it may comprisemodified amino acids, and it may be interrupted by non-amino acids. Theterms also encompass amino acid polymers that have been modified, forexample, via sulfonation, glycosylation, lipidation, acetylation,phosphorylation, iodination, methylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, ubiquitination, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” refers to either natural and/or unnatural or synthetic aminoacids, including glycine and both the D or L optical isomers, and aminoacid analogs and peptidomimetics.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site which specifically binds(“immunoreacts with”) an antigen. Structurally, the simplest naturallyoccurring antibody (e.g., IgG) comprises four polypeptide chains, twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. The immunoglobulins represent a large family of molecules thatinclude several types of molecules, such as IgD, IgG, IgA, IgM and IgE.The term “immunoglobulin molecule” includes, for example, hybridantibodies, or altered antibodies, and fragments thereof. It has beenshown that the antigen binding function of an antibody can be performedby fragments of a naturally-occurring antibody. These fragments arecollectively termed “antigen-binding units”. Antigen binding units canbe broadly divided into “single-chain” (“Sc”) and “non-single-chain”(“Nsc”) types based on their molecular structures.

Also encompassed within the terms “antibodies” are immunoglobulinmolecules of a variety of species origins including invertebrates andvertebrates. The term “human” as applies to an antibody or an antigenbinding unit refers to an immunoglobulin molecule expressed by a humangene or fragment thereof. The term “humanized” as applies to a non-human(e.g. rodent or primate) antibodies are hybrid immunoglobulins,immunoglobulin chains or fragments thereof which contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, rabbit or primate having thedesired specificity, affinity and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance and minimizeimmunogenicity when introduced into a human body. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody may also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

The term “stable” refers to compositions and compounds which havesufficient chemical stability to survive isolation from a reactionmixture to a useful degree of purity for use in a desired application.

The terms “fluorescent group”, “fluorophore”, “dye” or “fluorescentgroup” refer interchangeably to molecules, groups or radicals which arefluorescent. The term “fluorescent” as applied to a molecule of compoundis used to refer to the property of the compound of absorbing energy(such as UV, visible or IR radiation) and re-emitting at least afraction of that energy as light over time. Fluorescent groups,compounds or fluorophores include, but are not limited to discretecompounds, molecules, proteins and macromolecular complexes.Fluorophores also include compounds that exhibit long-lived fluorescencedecay such as lanthanide ions and lanthanide complexes with organicligand sensitizers.

A “subject” as used herein refers to a biological entity containingexpressed genetic materials. The biological entity is in variousembodiments, a vertebrate. In some embodiment, the biological entity isa mammal. In other embodiments, the subject is a biological entity whichcomprises a human.

A “control” is an alternative subject or sample used in an experimentfor comparison purposes. A control can be “positive” or “negative”. Forexample, where the purpose of the experiment is to detect adifferentially expressed transcript or polypeptide in cell or tissueaffected by a disease of concern, it is generally preferable to use apositive control (a subject or a sample from a subject, exhibiting suchdifferential expression and syndromes characteristic of that disease),and a negative control (a subject or a sample from a subject lacking thedifferential expression and clinical syndrome of that disease.

The term “FRET” refers to Foerster resonance energy transfer. In thepresent invention, FRET refers to energy transfer processes occurringbetween at least two fluorescent compounds, between a fluorescentcompound and a non-fluorescent component or between a fluorescentcomponent and a non-fluorescent component.

A “binding agent” is a molecule that exhibits binding selectivitytowards a binding partner or a target molecule to which it binds. Abinding agent may be a biomolecule such as a polypeptide such as anantibody or protein, polypeptide-based toxin, amino acid, nucleotide,polynucleotides including DNA and RNA, lipids, and carbohydrates, or acombination thereof. A binding agent may also be a hapten, drug,ion-complexing agent such as metal chelators, microparticles, syntheticor natural polymers, cells, viruses, or other fluorescent moleculesincluding the dye molecule according to the invention.

A “targeting moiety” is the portion of the binding agent that binds to abinding partner. A targeting moiety may be, without limitation, anucleotide sequence within a polynucleotide that selectively binds toanother polynucleotide or polypeptide. Another nonlimiting example of atargeting moiety may be a polypeptide sequence within a largerpolypeptide sequence which binds specifically to a polynucleotidesequence or a second polypeptide sequence. A targeting moiety may be asmall molecule or structural motif which will bind to a proteinreceptor, another small molecule motif, or complexing agent, withoutlimitation. The selective binding may be a specific binding event.

A “binding partner” is a molecule or particle which is bound by thetargeting moiety. It can be a cell, virus, fragment of a cell, antibody,fragment of an antibody, peptide, protein, polynucleotide, antigen,small molecule, or a combination thereof. It may be bound selectively orspecifically by the binding agent.

The term “signal to noise ratio” of fluorescence as referred to hereinin the context of a polypeptide-antibody complex, is the ratio of(fluorescent signal from a complex comprising a polypeptide bound by aprimary antibody which in turn is bound to a binding agent labeled witha compound of the invention)/(fluorescent signal from a mixture of thepolypeptide, an isotype control primary antibody, and the labeledbinding agent).

“Degree of labeling” or “DOL” as used herein herein refers to the numberof dye molecules which are attached per target molecule (including butnot limited to polypeptide and polynucleotide). For example, a singledye molecule per a polypeptide such as an antibody represents a 1.0degree of labeling (DOL). If more than one dye molecule, on average,reacts with and is crosslinked to a polypeptide such as an antibody, thedegree of labeling is greater than I and may further be a number otherthan a whole integer. The higher the number of DOL, the greater extentof labeling.

“Intracellular” as used herein refers to the presence of a givenmolecule in a cell. An intracellular molecule can be present within thecytoplasm, attached to the inner cell membrane, on the surface of anorganelle, or within an organelle of a cell.

“Substrate” or “solid substrate” when used in the context of a reactionsurface refers to the material that certain interaction is assayed. Forexample, a substrate in this context can be a surface of an array or asurface of microwell. It may also be a solid such as a polymer whichdoes not form a specific shape but has attachment points on its surface.

The terms “wavelength of maximum excitation” and “maximal fluorescenceexcitation wavelength” are used herein interchangeably. These termsrefer to the maximum wavelength at which a fluorescent compound absorbslight energy which excites the dye to emit maximal fluorescence. Theterm “absorption maximal wavelength” as applied to a dye refers thewavelength of light energy at which the dye most effectively absorbs. Afluorescent dye has a “maximal fluorescence emission wavelength” whichis the wavelength at which the dye most intensely fluoresces. When asingle wavelength is referred to for any dye, it refers to the maximalwavelength of excitation, absorption, or emission, according to thecontext of the term, for example, an absorption wavelength refers to thewavelength at which the compound has maximal absorption, and an emissionwavelength refers to the wavelength at which the dye most intenselyfluoresces.

Compounds of the Invention:

In one aspect, the invention provides a compound of Formula I:

wherein A is —O— or —NR₂—, where R₂ is —H or alkyl;

-   -   each R₁ and R₁′ is independently —H or alkyl, where each alkyl        may be the same or different and where any pair of two alkyl        groups may combine to form a cyclic ring;    -   a is an integer between 2 and 20;    -   L is a bond or a covalent linker comprising between 1 and 100        atoms; and    -   R_(x) is a reactive group capable of forming a covalent bond        upon reacting with a reaction substrate.

In one embodiment, A is —O—. In another embodiment, A is —NR₂—, forexample —NH₂, or —NH(alkyl)-.

R₁ and R₁′ are independently H or alkyl. In one embodiment, both R₁ andR₁′ are H. In another embodiment, at least one R₁ group is alkyl.

The variable “a” is an integer between 2 and 20. For example, “a” may be2, 3, 4, 5, 10, 15, or 20. When each R₁ and R₁′ are H, “a” representsthe number of repeating methylene units in A.

In selected embodiments,

is any of the groups shown below:

Linking moieties (generally represented by “L”) may be a bond or anygroup containing about 1 to 100 atoms. Synthetic accessibility andconvenience may generally dictate the nature of each linking moiety. Insome embodiments, a linking moiety is a group containing about 1-100atoms and formed of one or more chemical bonds selected such that thegroup is a stable moiety. In other embodiments, a linking moiety isformed of one or more carbon-hydrogen, carbon-nitrogen, carbon-oxygen,carbon-sulfur, carbon-phosphorus, nitrogen-hydrogen, sulfur-hydrogen,phosphorus-hydrogen, sulfur-oxygen, sulfur-nitrogen, sulfur-phosphorus,phosphorus-oxygen, phosphorus-nitrogen and oxygen-nitrogen bonds,wherein such bonds may be single, double, triple, aromatic andheteroaromatic bonds selected such that the linking moiety is stable. Alinking moiety can be, for example, a divalent alkyl radical.Alternatively, a linking moiety may be an alkyl group comprisingadditional ether, amine, amide, ester, sulfonyl, thioether, carboxamide,sulfonamide, hydrazide or morpholino, aryl and heteroaryl groups.

Linking moieties are generally formed of about 1-100 atoms. In someembodiments, linking moieties are formed of 1-50 non-hydrogen atoms aswell as additional hydrogen atoms. Such atoms may be, for example, C, N,O, P or S. In other embodiments, a linker moiety connecting two groupscomprises 1 to 50 consecutive bonds between the groups. Some linkermoieties may have 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 25,or 5 to 20 such consecutive bonds.

Non-limiting exemplary linking moieties are illustrated below:

In the above image, n represents a number of repeating methylene unitswhich can be varied such as to provide a desired length of the linker.Typically, n ranges from 1 to about 50. Some linkers will have an n of 1to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 5 to 30, 5 to 20, or 5 to 15.

Compounds of the invention comprise at least one R_(x) which is areactive group. A reactive group is a chemical moiety capable ofreacting with a reaction partner on a substrate or substrate molecule toform a covalent bond. A compound of the invention can be used to label awide variety of molecules or substrates that contain a suitable reactionpartner or are derivatized to contain a suitable reaction partner.“Reactive group” and “reaction partner” may refer to groups on acompound of the present invention, or to groups on a molecule to belabeled. Here, by way of convenience, but not limitation, a bond-forminggroup on a compound will generally be referred to as a reactive groupand a bond-forming group on the substrate molecule will generally bereferred to as a reaction partner. “Reaction substrate”, “substrate” and“reaction partner” are used interchangeably throughout this document.

The reactive group and its reaction partner may be an electrophile and anucleophile, respectively, that can form a covalent bond with or withouta coupling agent or catalyst. According to one embodiment, the reactivegroup is a photoactivatable group capable of reacting with a hydrocarbonmolecule upon ultraviolet photoactivation or photolysis. According toanother embodiment, the reactive group is a dienophile capable ofreacting with a conjugated diene via a Diels-Alder reaction. Accordingto yet another embodiment, the reactive group is a 1,3-diene capable ofreacting with a dienophile. According to still another embodiment, thereactive group is an alkyne capable of reacting with an azido functionalgroup to form a 1,2,3-triazole linkage. According to still anotherembodiment, the reactive group is a 2-(diphenylphosphino)benzoic acidmethyl ester capable of reacting with an azido functional group to forman amide linkage via so-called Staudinger reaction. Merely by way ofexample, examples of useful reactive groups, functional groups, andcorresponding linkages according to the present invention are listedbelow in Table 1.

TABLE 1 Examples of Reactive Groups, Functional Groups, and CovalentLinkages Reaction Resulting Covalent Reactive Group Partner/SubstrateLinkage activated esters* amines/anilines Carboxamides acrylamidesThiols Thioethers acyl azides** amines/anilines Carboxamides acylhalides amines/anilines Carboxamides acyl halides Alcohols/phenolsEsters acyl nitriles Alcohols/phenols Esters acyl nitrilesamines/anilines Carboxamides aldehydes amines/anilines Imines aldehydesor ketones Hydrazines Hydrazones aldehydes or ketones HydroxylaminesOximes alkyl halides amines/anilines alkyl amines alkyl halides ThiolsThioethers alkyl halides alcohols/phenols Esters alkyl sulfonates ThiolsThioethers alkyl sulfonates carboxylic acids Esters alkyl sulfonatesalcohols/phenols Esters anhydrides alcohols/phenols Esters anhydridesamines/anilines Carboxamides aryl halides Thiols Thiophenols arylhalides Amines aryl amines aziridines Thiols Thioethers boronatesGlycols boronate esters epoxides Thiols Thioethers haloacetamides ThiolsThioethers halotriazines amines/anilines Aminotrizaines halotriazinesalcohols/phenols triazinyl ethers imido esters amines/anilines Amidinesisocyanates amines/anilines Ureas isocyanates alcohols/phenols Urethanesisothiocyanates amines/anilines Thioureas maleimides Thiols Thioethersphosphoramidites Alcohols phosphite esters silyl halides Alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate estersThiols Thioethers sulfonate esters Alcohols Ethers sulfonyl halidesamines/anilines Sulfonamides sulfonyl halides phenols/alcohols sulfonateesters azide alkyne 1,2,3-triazole Cis-platinum guanosinePlatinum-guanosine complex *Activated esters, as understood in the art,generally have the formula —COLg, where Lg is a good leaving group, suchas succinimidyloxy (—OC₄H₄O₂), sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H), or-1-oxybenzotriazolyl (—OC₆H₄N₃), for example; or an aryloxy group oraryloxy substituted one or more times by electron-withdrawingsubstituent(s), such as nitro, fluoro, chloro, cyano, trifluoromethyl,or combinations thereof, for example, used to form activated arylesters; or a carboxylic acid activated by a carbodiimide to form ananhydride or mixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a)and R^(b), which may be the same or different, are independently C₁-C₆alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl,3-dimethylaminopropyl, or N-morpholinoethyl. **Acyl azides can alsorearrange to isocyanates.

The reactive group may be one that will react with an amine, a thiol, ahydroxyl or an aldehyde. The reactive group may be an amine-reactivegroup, such as a succinimidyl ester (SE), for example, or athiol-reactive group, such as a maleimide, a haloacetamide, or amethanethiosulfonate (MTS), for example, or an aldehyde-reactive group,such as an amine, an aminooxy, or a hydrazide, for example.

One of skills can appreciate that an ionic group requires a counter ionto balance its charge. For example, negatively charged —SO₃ ⁻ groups maynecessitate cations to balance the negative charge. Likewise, apositively charged ammonium may require an anion to maintain neutrality.In general, the nature of the counter ion is not critical as long as thecounter ion does not lower the solubility of said fluorescent group. Insome embodiments, when an anion is —SO₃ ⁻, the counter ion is H⁺, Na⁺,K⁺ or an ammonium group. If additional fluorescent compounds are used,such fluorescent compounds may intrinsically possess a positive chargeor negative charge. In such a case, the intrinsic charge may act as acounter ion. Alternatively, the intrinsic charge may require a counterion for maintaining neutrality. The rule for selecting a counter ion forany intrinsic charge is as previously described. In some embodiments ofthe invention, at least one sulfonate group is present (—SO₃ ⁻) and anynecessary counter ion is selected from H⁺, Na⁺, K⁺ and an ammonium. Forreason of simplicity, any dissociable counter ion or counter ions formost of the fluorescent group structures depicted herein may not beshown.

Exemplary structures of specific compounds of the invention are shown inTable 2 below:

TABLE 2 Exemplary compounds of Formula I Abs/Em (nm) Compound Structure(in PBS) Cascade Blue acetyl azide

396/410 (MeOH) Alexa Fluor 405

399/425 1

403/438 2

404/433 2 (streptavidin conjugate)

404/431 2 (goat anti- mouse lgG conjugate)

404/431 3

455/522 4

455/522 5

455/522 6

455/522Table 2 shows exemplary compounds of the invention. Compounds 1-3 aredesigned to show maximum excitation around 405 nm, while compounds 3-5show maximum excitation about 470 nm.

(1) Uses of the Subject Compounds.

The subject compounds find use in a variety of different applications.One application of interest is the use of the subject compounds aslabeling agents which are capable of imparting a fluorescent property toa particular composition of matter. The compounds of the presentinvention can be used to react with any of a broad range of molecules,including but not limited to, biomolecules such as polypeptides,polypeptide-based toxins, amino acids, nucleotides, polynucleotidesincluding DNA and RNA, lipids, and carbohydrates, and any combinationsthereof. Additionally, the compounds of the invention can be used toreact with haptens, drugs, ion-complexing agents such as metalchelators, microparticles, synthetic or natural polymers, cells,viruses, other fluorescent molecules including the dye moleculeaccording to the invention, or surfaces. The substrate moleculestypically comprise one or more functional groups, which react with thereactive group of the subject compounds to form covalent or non-covalentlinkage. In one aspect, the reactive group of a compound of theinvention is an activated ester (such as a succinimidyl ester, or SE), amaleimide, a hydrazide or an aminooxy group. Accordingly, in someaspects, functional group from a substrate molecule (or reactionsubstrate) is an amine, a thiol, an aldehyde or ketone. The resultingfluorescently labeled substrate molecules may be referred to asconjugates or labeled substrate molecules. Any methods practiced in theart (e.g., Brinkley, Bioconjugate Chem. 3, 2(1992), incorporated hereinby reference) for preparing fluorescent group-substrate conjugates areapplicable for practicing the subject invention.

Conjugates of biomolecules and compounds of the invention usually havehigh fluorescence yield while typically retaining the criticalparameters of unlabeled biomolecules, such as solubility, selectivebinding to a receptor or nucleic acid, activation or inhibition of aparticular enzyme or the ability to incorporate into a biologicalmembrane. Nevertheless, conjugates with the highest degree of labelingmay still precipitate or bind nonspecifically. As necessary, aless-than-maximal degree of labeling may be acceptable in order topreserve function or binding specificity. Preparing the conjugates ofthe invention may involve experimentation to optimize properties.Following conjugation, unconjugated labeling reagent may be removed bytechniques known in the art such as by gel filtration, dialysis,conjugate precipitation and resolubilization, HPLC or a combination ofthese techniques. The presence of free dye, particularly if it remainschemically reactive, may complicate subsequent experiments with thebioconjugate.

Nucleic Acids

In another embodiment, the subject compounds can be used to conjugatewith a nucleoside, a nucleotide, or a polynucleotide, wherein any ofsuch molecules may be natural or synthetic, modified or unmodified. Thecompound of the invention used for labeling may comprise a reactivegroup which is a phosphoramidite, an activated ester (such as asuccinimidyl ester), an alkylating group or a reactive platinum complex.Such molecules may contain or are derivatized to contain one or morereaction partners for the reactive groups on the compounds of theinvention. A reactive group of a compound of the invention may reactwith a suitable reaction partner on said molecule to form a covalentlinkage. For example, a phosphoramidite group may react with a hydroxylgroup to form a phosphate linkage after deprotection; a succinimidylester or the like may react with an amine group to form an amidelinkage; and a reactive platinum complex may react with a guanosine baseto form a platinum complex linkage. In one embodiment, a reactivecompound of the invention comprising an activated ester is reacted witha nucleotide triphosphate comprising a base comprising an aminoalkynylgroup, an aminoallyl group or an aminoalkyl group to form afluorescently labeled nucleotide triphosphate. Such a labeled nucleotidetriphosphate is often used to prepare a fluorescently labeled nucleicacid polymer via enzymatic incorporation.

In some embodiments, the fluorescent compound of the invention isreacted with a group or linker attached to the C-5 position of a uridineor cytidine residue. This position is not involved in Watson-Crickbase-pairing and interferes little with hybridization to complementarysequences. An aminoalkynyl linker may be introduced between afluorescent moiety and the nucleotide in order to reduce fluorophoreinteraction with enzymes or target binding sites. In addition to thisfour-atom bridge, seven- to 10-atom spacers may be introduced thatfurther separate the fluorophore from the base. The use of longerspacers may result in brighter conjugates and increased haptenaccessibility for secondary detection reagents.

Alternatively, deoxycytidine triphosphates may be prepared which aremodified at the N-4 position of cytosine using a 2-aminoethoxyethyl(OBEA) linker. Possible steric interference caused by the presence ofthe fluorescent fluorophore may be reduced by the use of additionalspacers.

Fluorescently labeled DNA may be prepared from a fluorescently labelednucleotide triphosphate by PCR reaction, terminal transferase-catalyzedaddition or nick translation. Various polymerases may be used in suchreactions. Such polymerases include Taq polymerase (useful e.g. inpolymerase chain reaction (PCR) assays), DNA polymerase I (useful e.g.in nick-translation and primer-extension assays), Klenow polymerase(useful e.g. in random-primer labeling), Terminal deoxynucleotidyltransferase (TdT) (useful e.g. for 3′-end labeling), Reversetranscriptase (e.g. for synthesizing DNA from RNA templates) or otherpolymerases such as SP6 RNA polymerase, T3 RNA polymerase and T7 RNApolymerase for in vitro transcription.

Alternatively, a fluorescently labeled nucleic acid polymer may beprepared by first enzymatically incorporating an amine-labelednucleotide into a nucleic acid polymer to result in an amine-labelednucleic acid polymer, followed by the labeling of said amine-labeledpolymer with a compound of the invention. More information on thepreparation and use of fluorescently labeled nucleotide triphosphatescan be found in U.S. Pat. Nos. 4,711,955 and 5,047,519. Stillalternatively, a nucleic acid polymer, such as a DNA, may be directlylabeled with a compound of the invention comprising a reactive platinumcomplex as the reactive group, wherein the platinum complex form acoordinative bond with a nitrogen atom of a guanosine base such asdescribed in U.S. Pat. No. 5,714,327.

Aminoacids and Polypeptides

In another embodiment, the subject compounds can be used to conjugatewith an amino acid, amino acid analog or a polypeptide. Labeledaminoacids, amino acid analogs and polypeptides may be labeled byreacting the compounds of the invention with amino acids, amino acidanalogs and polypeptides comprising reaction partners for the reactivegroups on said compounds. Such reaction partners may be natural orunnatural groups present in said polypeptides. By way of example,reaction partners may be the natural residues such as amino groups,which are part of natural lysine residues, or thiol groups, which arepart of natural cysteine groups.

In order to achieve the maximal fluorescence possible, a protein may belabeled with as many molecules of the same fluorescent group aspossible, to the degree that the biological activity of the protein isminimally affected by the labeling. In other cases it may be desirableto avoid fluorescence quenching resulting from multiple fluorescentgroup molecules on the protein interacting with each other. Dye-dyeinteractions may be physical, such as dye aggregation, or may be aspectral, such as FRET-based energy transfer, or a combination of both.Either type of interaction may lead to fluorescence quenching, which canbe characterized by a slow rise and then a rapid drop of the totalfluorescence of the labeled protein as the degree of labeling increases.A primary reason for fluorescence quenching of a labeling fluorescentgroup on protein is believed to be due to formation of dye aggregatessuch as dye dimer. When dye dimer formation occurs, the absorptionspectrum of the fluorescent group-protein conjugate typically show adoublet peak.

In some embodiments, the complexes of the methods of the invention havea signal-to-noise ratio that is equal or greater than about 70, about80, about 90, about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 255, about 260,about 265, about 270, about 275, about 280, about 285, about 290, about295, about 300, about 305, about 310, about 315, about 320, about 330,about 340, about 350, about 360, about 370, about 380, about 390, orabout 400. In some embodiments of the complexes of the methods of theinvention, the signal-to-noise ratio is no less than about 100, about110, about 120, about 130, about 140, about 150, about 160, about 170,about 180, about 190, about 200, about 210, about 220, about 230, about240, about 250, about 255, about 260, about 265, about 270, about 275,about 280, about 285, about 290, about 295, about 300, about 305, about310, about 315, about 320, about 330, about 340, about 350, about 360,about 370, about 380, about 390, or about 400.

(2) Uses of the Labeled Biomolecules of the Invention

The subject compounds provide an effective tool for labelingbiomolecules for a wide variety of applications. Labeling allows one todiscern interactions involving biomolecules such as proteins,glycoproteins, nucleic acids, and lipids, as well as inorganicchemicals, or any combinations thereof. The interactions may be betweennucleic acid molecules, between nucleic acid and protein, and betweenprotein and small molecules. The interactions may be discerned in acell-free biological system, in a cellular system (includingintracellular and extracellular systems), or in vivo, which encompasseswhich encompasses activities within a cell that is within a tissue ororgan or a subject Delineating the various interactions is often asignificant step in scientific research and development, drug design,screening and optimization, phylogenetic classification, genotypingindividuals, parental and forensic identification, environmentalstudies, diagnosis, prognosis, and/or treatment of disease conditions.

Biomolecules labeled according to the methods of the invention may beused as binding agents to detect their binding partners, the targets oftheir biological interaction, as described above. For example, a proteincan be labeled with a dye of the invention and used to bind to a cellsurface receptor. In some embodiments of the invention, a binding agentis labeled with a substituted cyanine dye having maximal fluorescenceexcitation wavelength of equal or greater than 660 nm, a water solublepolymer group, and a reactive group under conditions effective tocrosslink the dye and the binding agent. In some embodiments, thesubstituted cyanine dye is substituted by a non-spiro substituent. Abinding agent so labeled is contacted with its binding partner, and thefluorescent label is detected. In other embodiments, a binding agent isreacted with a compound of structure of Formula I, II, III, IV or Vunder conditions effective to crosslink the compound with the bindingagent

Labeled molecules of the invention may be used as part of FRET pairs in,a variety of biological assays and methods, whether as donor or acceptormolecules. A person skilled in the art will know to select a suitableFRET partner based on the specific application. Such applicationsinclude, but are not limited to, assays involving molecular beacons,FRET protease assays, flow cytometry, nucleic acid hybridization and anyother applications where the relative spatial localization of two ormore moieties must be probed. FRET is generally useful on scales of 10to 100 Å. In one embodiment, both the donor and the acceptor of a FRETpair are labeled molecules of the invention. In another embodiment, onemember of a FRET pair is a labeled oligonucleotide of the inventionwhich is capable of annealing to a complementary oligonucleotide labeledwith a second member of the FRET pair, such that annealing leads to anincrease in the efficiency of energy transfer. In this example, thesecond member of the FRET pair may be a fluorophore of the invention ormay be a different fluorophore.

Such fluorophores include Acridine orange, Acridine yellow, Alexa Fluorfluorescent groups, ATTO fluorescent groups, Bodipy fluorescent groups,Auramine O, Benzanthrone, 9,10-Bis(phenylethynyl)anthracene,5,12-Bis(phenylethynyl)naphthacene, Carboxyfluorescein diacetate,Calcein, Carboxyfluorescein, 1-Chloro-9,10-bis(phenylethynyl)anthracene,2-Chloro-9,10-bis(phenylethynyl)anthracene, Coumarin, Cyanine, Cy2, Cy3,Cy3.5, Cy5, Cy5.5, Cy7, DyLight Fluor fluorescent groups, Fluorescein,2′,7′-dichlorodihydrofluorescein, Hilyte Fluor fluorescent groups, LDS751, Oregon Green, Perylene, Phycobilin, Phycoerythrin,Phycoerythrobilin, Pyrene, Rhodamine and Ruthenium(II)tris(bathophenanthroline disulfonate). These compounds and derivativesor radicals of these compounds may be used as fluorophores.

Other examples of fluorescent groups which may be used include but arenot limited to 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonicacid, acridine and derivatives such as acridine and acridineisothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate(Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide,BODIPY™ and its derivatives and analogs, Brilliant Yellow, cyaninefluorescent groups such as Cy3 and Cy5 and other derivatives, coumarinand derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC,Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),fluorescein and derivatives such as 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;4-methylumbelliferone, oxazine fluorescent groups such as Nile Blue andother analogs; pyrene and derivatives such as pyrene, pyrene butyrateand succinimidyl 1-pyrene butyrate; rosamine fluorescent groups,tetramethyl rosamine, and other analogs, rhodamine and derivatives suchas 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC) and thiazine fluorescentgroups such as methylene blue and analogs. Additional fluorophoresapplicable for use in the present are disclosed in US patent applicationNos. 2003/0165942, 2003/0045717, and 2004/0260093 and U.S. Pat. No.5,866,366 and WO 01/16375, both of which are incorporated herein byreference. Additional examples are described in U.S. Pat. No. 6,399,335,published U.S. patent application No. 2003/0124576, and The Handbook—‘AGuide to Fluorescent Probes and Labeling Technologies, Tenth Edition’(2005) (available from Invitrogen, Inc./Molecular Probes), all of whichare incorporated herein by reference.

Fluorescent compounds for use in the invention may also includefluorescent proteins. Such fluorescent proteins known in the art includeGFP and its various derivatives, described e.g. in U.S. Pat. Nos.5,625,048; 5,777,079; 6,066,475; 6,319,669; 6,046,925; 6,124,128 and6,077,707. Additional fluorescent proteins are Y66F, Y66H, EBFP, GFPuv,ECFP, AmCyan1, Y66W, S65A, S65C, S65L, S65T, EGFP, ZsGreen1, EYFP,ZsYellow1, DsRed, DsRed2, AsRed2, mRFP1 and HcRed1.

Many such fluorescent groups are commercially available and may be usedin the synthesis of compounds of the present invention. Commercialsources of reactive fluorescent groups include Invitrogen (MolecularProbes), AnaSpec, Amersham (AP Biotech), Atto-Tec, Dyomics, Clontech andSigma-Aldrich.

In some applications, it is desirable to quench the labeled molecules ofthe invention. A variety of quenchers known in the art may be used.Non-limiting examples include Black Hole Quencher™ moieties, DABCYL,Reactive Red 4 (Cibacron Brilliant Red 3B-A), Malachite Green,4-Dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), and4,4′-Diisothiocyanaitodihydro-stilbene-2,2′-disulfonic acid. By way ofexample, a molecular beacon may be labeled with a compound of theinvention as well as with a suitable quencher. In the closedconformation of the beacon, the fluorophore is quenched. When the beaconopens as a result of a recognition or binding event, the fluorescence ofthe fluorophore increases significantly.

In still another embodiment, the invention provides an energy transferfluorescent group comprising a first donor fluorescent group and secondacceptor fluorescent group wherein: the donor fluorescent group andacceptor fluorescent group are covalently linked to form a FRET pair; atleast one of the donor fluorescent group and acceptor fluorescent groupis a fluorescent group of the invention; and the energy transferfluorescent group optionally comprises a reactive group. Methods forpreparing energy transfer fluorescent groups and uses thereof have beenpreviously described. See U.S. Pat. No. 6,479,303 and WO 00/13026.

In one embodiment, a fluorescent group of the invention is used to labela fluorescent protein to form a so-called tandem dye, wherein thefluorescent group of the invention and the fluorophore of thefluorescent protein form an energy transfer pair (i.e., FRET pair). Insuch a FRET pair, the fluorescent group of the invention is either thedonor fluorescent group or the acceptor fluorescent group and, likewise,the fluorophore of the protein is either the acceptor fluorescent groupor the donor fluorescent group, such that the FRET pair can be excitedat or near the absorption maxima of the donor fluorescent group and thefluorescence collected at the emission maxima of the acceptorfluorescent group, resulting in a large Stokes shift. Suitablefluorescent proteins for preparing tandem dyes include, but are notlimited to, various phycobiliproteins such as Allophycocyanin B,Allophycocyanin (APC), C-Phycocyanin, R-Phycocyanin, Phycoerythrocyanin,C-Phycoerythrin, b-Phycoerythrin, B-Phycoerythrin, R-Phycoerythrin(R-PE), and the likes. Phycobiliproteins are proteins comprising bilinas prosthetic groups, which are also the fluorophores of the proteins.Preferably, the phycobiliproteins are R-PE or APC. To achieve suitableFRET efficiency, one may choose a fluorescent group of properwavelengths so that the emission of the donor fluorescent group and theabsorption of the acceptor fluorescent group have sufficient spectraloverlap. Detailed methods for fluorescent group selection and forpreparing tandem dyes are disclosed in U.S. Pat. Nos. 4,520,110 and5,714,386. Dyes of the invention may also be useful forfluorescence-activated cell sorting (FACS) or flow cytometry studies.Commercial flow cytometers are typically equipped with 1 to 3 excitationlight sources, more commonly 1 to 2 excitation light sources. Thus, inorder to detect multiple targets, each target may be stained with adifferent fluorescent group having a different emission and thedifferent fluorescent groups all need to be efficiently excited by acommon excitation source.

In one embodiment, a compound of the invention is applied to abiological sample comprising a plurality of polypeptides and optionallyother biological molecules under a condition facilitating the covalentlabeling of said polypeptides. In some embodiments, the reactive groupof the compound is an activated ester, a maleimide, an iodoacetamide, abromoacetamide, a hydrazide, an amine or an aminooxy group. Thebiological sample may be a cell lysate or a tissue lysate. The resultinglabeled polypeptides or cellular components may be analyzed and/orpurified by any of a variety of known tools or techniques, including,but not limited to, protein microarrays, chromatography and gelelectrophoresis.

The present invention also provides kits comprising compounds of theinvention and/or fluorescent group-substrate conjugates of the inventionfor various assays as selectively described above. A kit of theinvention may comprise one or more compounds of the invention andinstructions instructing the use of said compound. For example, a kitmay comprise one or more compounds of the invention for labeling asubstrate, one or more buffers for the labeling reaction and productpurification, a chromatography column for purifying the resultingfluorescent group-substrate conjugate, a protocol for carrying out theprocedure, optionally any additional reagents and optionally anyreference standard. In another embodiment, a kit comprises one or morefluorescent group-substrate conjugates of the invention, one or morebuffers, a protocol for the use of said conjugate(s), optionally anyother reagents for an assay, and optionally any calibration standard(s).The kit may further contain other materials or devices of use inpurifying the conjugation products.

The signals produced by the fluorescent groups of the invention may bedetected in a variety of ways. Generally, a change of signal intensitycan be detected by any methods known in the art and is generallydependent on the choice of fluorescent group used. It can be performedwith the aid of an optical system. Such system typically comprises atleast two elements, namely an excitation source and a photon detector.Numerous examples of these elements are available in the art. Anexemplary excitation source is a laser, such as a polarized laser. Thechoice of laser light will depend on the fluorescent group attached tothe probe. For most of the fluorescent groups, the required excitationlight is within the range of about 300 nm to about 1200 nm, or morecommonly from about 350 nm to about 900 nm. Alternatively, compounds ofthe invention may be excited using an excitation wavelength of about 200to about 350 nm, 350 to 400 nm, 400 to 450 nm, 450 to 500 nm, merely byway of example. Those skilled in the art can readily ascertain theappropriate excitation wavelength to excite a given fluorophore byroutine experimentation, (see e.g., The Handbook—‘A Guide to FluorescentProbes and Labeling Technologies, Tenth Edition’ (2005) (available fromInvitrogen, Inc./Molecular Probes) previously incorporated herein byreference). Where desired, one can employ other optical systems. Theseoptical systems may comprise elements such as optical reader,high-efficiency photon detection system, photo multiplier tube, gatesensitive FET's, nano-tube FET's, photodiode (e.g. avalanche photodiodes (APD)), camera, charge couple device (CCD), electron-multiplyingcharge-coupled device (EMCCD), intensified charge coupled device (ICCD),and confocal microscope. These optical systems may also comprise opticaltransmission elements such as optic fibers, optical switches, mirrors,lenses (including microlens and nanolens), collimators. Other examplesinclude optical attenuators, polarization filters (e.g., dichroicfilter), wavelength filters (low-pass, band-pass, or high-pass),wave-plates, and delay lines. In some embodiments, the opticaltransmission element can be planar waveguides in optical communicationwith the arrayed optical confinements. See, e.g., U.S. Pat. Nos.7,292,742, 7,181,122, 7,013,054, 6,917,726, 7,267,673, and 7,170,050.These and other optical components known in the art can be combined andassembled in a variety of ways to effect detection of distinguishablesignals.

Fluorescently labeled polynucleotides of the invention find use in avariety of applications. Such applications can involve interactionsbetween nucleic acids, e.g., interactions between DNA and DNA, DNA andRNA, and RNA and RNA, or any other non-naturally occurring nucleic acidsPNA, LNA, and/or TNA. Various applications can also involve interactionsbetween nucleic acids and proteins, lipids or combinations thereof.Non-limiting examples of specific nucleic acid assays include nucleicacid amplification, both quantitative or end-point amplification,hybridization in solution or on a substrate (e.g., array hybridization),gel shifts, and nucleic acid sequencing. The fluorescently labeledpolynucleotides can be used in solution phase or immobilized on asubstrate.

In one embodiment, the labeled polynucleotides are used as hybridizationprobes. One application of hybridization probes is fluorescent in situhybridization (FISH). In this technique, a labeled polynucleotidecomplementary to a sequence of interest is annealed to fixed chromosomespreparations, and the presence of the sequence of interest as well asthe chromosomal localization is detected by microscopy. FISH can beperformed by immobilizing the nucleic acids of interest on a substrateincluding without limitation glass, silicon, or fiber. FISH may also beused quantitatively (Q-FISH) to detect the presence and length ofrepetitive sequences such as telomeres. This may be done by quantitatingthe intensity of emitted fluorescence as measured by microscopy. FISHassays utilizing the subject fluorescent compounds can be performed fordetecting a specific segment of a DNA molecule or a chromosome. Thesefeatures can be used in genetic counselling (e.g., prenatal-screens),medicine, and species identification.

In some embodiments, labeled polynucleotides can be used as primers inamplification reactions such as PCR. In yet another embodiment, acompound of the invention may be used to label a polynucleotide which issubsequently used as a probe may be a hybridization probe or a real-timePCR probe. Such a probe may be labeled with a second fluorescent groupto form a FRET pair with the first fluorescent group of the invention.Methods for the preparation and use of PCR probes are well known to oneskilled in the art.

In one embodiment of the invention, a method is provided for detectingor quantifying a target nucleic acid, the method comprising the stepsof: a) providing a labeled polynucleotide (“probe”) of the presentinvention; b) contacting said labeled polynucleotide with the nucleicacid target so as to allow for hybridization of the probe with thenucleic acid target; and c) detecting or quantifying said nucleic acidtarget by measuring a change in the fluorescence of the probe upon thehybridization of the nucleic acid probe with the nucleic acid target.

As used herein, hybridization occurs when the probe form a complex withthe target nucleic acid. In general, the complex is stabilized, at leastin part, via hydrogen bonding between the bases of the nucleotideresidues. The hydrogen bonding may occur by Watson-Crick base pairing,Hoogstein binding, or in any other sequence-specific manner.Hybridization may constitute a step in a more extensive process, such asthe initiation of a PCR reaction, or the enzymatic cleavage of apolynucleotide by a ribozyme.

After hybridization between the probe and the target has occurred, achange in the intensity of the fluorescence of the probe may bemeasured. Such change before and after hybridization can yield apositive gain or negative reduction in the detected signal intensity.Depending on the specific hybridization assay that is run, more than oneevent after hybridization may contribute to the generation of a changein signal intensity. For example, an increase in reporter signal mayresult by way of spatial extension or separation of the reporterfluorescent group from the quencher group while both are still attachedto the probe. In addition, either the reporter or the quencher of theprobe can be separated by way of cleavage via an enzyme (e.g., apolymerase having a 5′ to 3′ exonuclease), thereby generating a reportersignal that is detected. As noted above, both the reporter and thequencher are defined in functional terms, such that these groups can beidentical though serving, relative to each other, a different functionwhen used in a hybridization reaction. For example, a group attached toa probe is a quencher because it reduces the emission of an opticalsignal when the probe is not hybridized with the target nucleic acid(typically when the probe assumes a random state). The same group canbecome a reporter fluorescent group upon being cleaved by an enzymeafter hybridization with the target nucleic acid as the signal of thefluorescent group is now detected during the assay.

The signal detection methods described previously can be applied tonucleic acid amplification in which the target nucleic acid is increasedin copy number. Such increase may occur in a linear or in an exponentialmanner. Amplification may be carried out by natural or recombinant DNApolymerases such as Taq polymerase, Pfu polymerase, T7 DNA polymerase,Klenow fragment of E. coli DNA polymerase, Tma DNA polymerase, exo-TliDNA polymerase, exo-KOD DNA polymerase, exo-JDF-3 DNA polymerase,exo-PGB-D DNA polymerase, UlTma (N-truncated) Thermatoga martima DNApolymerase, Sequenase, and/or RNA polymerases such as reversetranscriptase.

A preferred amplification method is polymerase chain reaction (PCR).General procedures for PCR are taught in U.S. Pat. No. 4,683,195(Mullis) and U.S. Pat. No. 4,683,202 (Mullis et al.). Briefly,amplification of nucleic acids by PCR involves repeated cycles ofheat-denaturing the DNA, annealing two primers to sequences that flankthe target nucleic acid segment to be amplified, and extending theannealed primers with a polymerase. The primers hybridize to oppositestrands of the target nucleic acid and are oriented so that thesynthesis by the polymerase proceeds across the segment between theprimers, effectively doubling the amount of the target segment.Moreover, because the extension products are also complementary to andcapable of binding primers, each successive cycle essentially doublesthe amount of target nucleic acids synthesized in the previous cycle.This results in exponential accumulation of the specific target nucleicacids at approximately a rate of 2″, where n is the number of cycles.

A typical conventional PCR thermal cycling protocol comprises 30 cyclesof (a) denaturation at a range of 90° C. to 95° C. for 0.5 to 1 minute,(b) annealing at a temperature ranging from 50° C. to 65° C. for 1 to 2minutes, and (c) extension at 68° C. to 75° C. for at least 1 minute.Other protocols including but not limited to universal protocol as wellas fast cycling protocol can be performed the subject probes as well.

A variant of the conventional PCR is a reaction termed “Hot Start PCR”.Hot Start PCR techniques focus on the inhibition of polymerase activityduring reaction preparation. By limiting polymerase activity prior toPCR cycling, non-specific amplification is reduced and the target yieldis increased. Common methods for Hot Start PCR include chemicalmodifications to the polymerase (see, e.g., U.S. Pat. No. 5,773,258),inhibition of the polymerase by a polymerase-specific antibody (see,e.g., U.S. Pat. No. 5,338,671), and introduction of physical barriers inthe reaction site to sequester the polymerase before the thermal cyclingtakes place (e.g., wax-barrier methods). The reagents necessary forperforming Hot Start PCR are conveniently packaged in kits that arecommercially available (see, e.g., Sigma's JumpStart Kit).

Another variation of the conventional PCR that can be performed with thesubject probes is “nested PCR” using nested primers. The method ispreferred when the amount of target nucleic acid in a sample isextremely limited for example, where archival, forensic samples areused. In performing nested PCR, the nucleic acid is first amplified withan outer set of primers capable of hybridizing to the sequences flankinga larger segment of the target nucleic acid. This amplification reactionis followed by a second round of amplification cycles using an inner setof primers that hybridizes to target sequences within the large segment.

The subject probes can be employed in reverse transcription PCR reaction(RT-PCR), in which a reverse transcriptase first coverts RNA moleculesto double stranded cDNA molecules, which are then employed as thetemplate for subsequent amplification in the polymerase chain reaction.In carrying out RT-PCR, the reverse transcriptase is generally added tothe reaction sample after the target nucleic acids are heat denatured.The reaction is then maintained at a suitable temperature (e.g., 30°C.-45° C.) for a sufficient amount of time (e.g., 5-60 minutes) togenerate the cDNA template before the scheduled cycles of amplificationtake place. Such reaction is particularly useful for detecting thebiological entity whose genetic information is stored in RNA molecules.Non-limiting examples of this category of biological entities includeRNA viruses such as HIV and hepatitis-causing viruses. Another importantapplication of RT-PCR embodied by the present invention is thesimultaneous quantification of biological entities based on the mRNAlevel detected in the test sample.

The subject probes can also be employed to perform ligase chainpolymerase chain reaction (LCR-PCR). The method involves ligating thetarget nucleic acids to a set of primer pairs, each having atarget-specific portion and a short anchor sequence unrelated to thetarget sequences. A second set of primers containing the anchor sequenceis then used to amplify the target sequences linked with the first setof primers. Procedures for conducting LCR-PCR are well known to artisansin the,field, and hence are not detailed herein (see, e.g., U.S. Pat.No. 5,494,810).

The subject probes are particularly suited for use in a homogeneousassay. In such an assay, a target nucleic acid is detected and/orquantified without the requirement of post-assay processing to recordthe result of the assay. For example, a homogeneous PCR reaction can becarried out in a closed sample holder (e.g., a tube, a sample capillaryor thermalchip), and no further addition or removal of reagents isnecessary to record the result once the assay is started. Homogeneousassays allow recordation of the result of the assay in real time. Wheredesired, in practicing the subject methods, the result of the assay canbe continuously recorded as the assay progresses in time or recordedintermittently at one or more point during the assay or upon completionof the assay.

Where desired, homogeneous assays can be multiplexed, i.e., more thanone target nucleic acid can be detected in one assay. In a multiplexassay, two or more specific nucleic acid probes, which differ in thenature of their covalently attached fluorescent groups, are added to themixture to be assayed. The fluorescent groups are chosen to producedistinguishable fluorescent signals from each specific nucleic acidprobe. The signals of the different fluorescent group combinations ofthe nucleic acid probes can be recorded simultaneously to detect and/orquantify the corresponding target nucleic acids. Multiplexing greatlyreduces the cost of analysis and can tremendously increase throughput inhigh volume settings.

The subject probes can be used to detect single mutations. Accordingly,methods are provided to use the probes of the invention to detect as fewas a single mismatch between the probe sequence and a target sequence.Such high specificity in nucleic acid detection by PCR is highlyvaluable in clinical diagnosis and genetic research. For example, manydiseases are associated with single mutations at different sites in thehuman genome. Although in theory this type of genetic variations, alsocalled single nucleotide polymorphism or SNP, may be detected bysequencing, such sequencing method is not expected to be practical on alarge scale due to high cost and low efficiency. Detection of SNP by anamplification reaction is feasible with the use of the subject probes.

The subject probes are also particularly suited for monitoring nucleicacid amplification reactions. In a related embodiment, the presentinvention provides a method of monitoring the increase in a targetnucleic acid during amplification of said target. The method typicallyinvolves a) providing an amplification reaction mixture that comprisessaid target nucleic acid, at least one primer that hybridizes to thetarget nucleic acid, a labeled oligonucleotide probe of the presentinvention that provides a detectable signal, the intensity of which isproportional to the increase in the target nucleic acid in theamplification; (b) treating said mixture under conditions for amplifyingsaid target nucleic acid; and (c) measuring the amount of said signalproduced by said mixture during said treating step (c). Where desired,the amount of signal is determined continuously throughout theamplification reaction or determined intermittently during theamplification reaction. The amplification can be exponentially with theuse of a primer pair or linearly with the use of one primer of the pair.

The increase in signal intensity during the amplification reaction maydue to the step of hybridization of the probe to the target nucleic acidand also the step of cleavage via the action of the polymerase utilizedin the amplification reaction.

In one aspect, the subject methods exploit the 5′ to 3′ nucleaseactivity of a polymerase when used in conjunction with PCR. When thesubject probe is added concomitantly with the primer at the start ofPCR, and the signal generated from hydrolysis of the labelednucleotide(s) of the probe provides a means for detection of the targetsequence during its amplification. Numerous polymerases are suited tocatalyze primer and template-dependent nucleic acid synthesis andpossess the 5′ to 3′ nuclease activity. Non-limiting examples includeDNA polymerases such as E. coli DNA polymerase I, Thermus thermophilus(Tth) DNA polymerase, Bacillus stearothermophilus DNA polymerase,Thermococcus littoralis DNA polymerase, and Thermus aquaticus (Taq) DNApolymerase. Where desired, temperature stable polymerases can beemployed in a nucleic acid amplification reaction. See, e.g., U.S. Pat.No. 4,889,818 that discloses a representative thermostable enzymeisolated from Thermus aquaticus. Additional representative temperaturestable polymerases include without limitation, e.g., polymerasesextracted from the thermostable bacteria Thermus flavus, Thermus ruber,Thermus thermophilus, Bacillus stearothermophilus (which has a somewhatlower temperature optimum than the others listed), Thermus lacteus,Thermus rubens, Thermotoga maritima, Thermococcus littoralis, andMethanothermus fervidus.

In another embodiment, nucleic acid amplification can be performed withpolymerases that exhibit strand-displacement activity (also known asrolling circle polymerization). Strand displacement can result in thesynthesis of tandem copies of a circular DNA template, and isparticularly useful in isothermal PCR reaction. Non-limiting examples ofrolling circle polymerases suitable for the present invention includebut are not limited to T5 DNA polymerase (Chatterjee et al., Gene97:13-19 (1991)), and T4 DNA polymerase holoenzyme (Kaboord andBenkovic, Curr. Biol. 5:149-157 (1995)), phage M2 DNA polymerase(Matsumoto et al., Gene 84:247 (1989)), phage PRD1 DNA polymerase (Junget al., Proc. Natl. Aced. Sci. USA 84:8287 (1987), and Zhu and Ito,Biochim. Biophys. Acta. 1219:267-276 (1994)), Klenow fragment of DNApolymerase I (Jacobsen et al., Eur. J. Biochem. 45:623-627 (1974)).

A preferred class of rolling circle polymerases utilizes protein primingas a way of initiating replication. Exemplary polymerases of this classare modified and unmodified DNA polymerase, chosen or derived from thephages Φ29, PRD1, Cp-1, Cp-5, Cp-7, Φ15, Φ1, Φ21, Φ25, BS 32 L17, PZE,PZA, Nf, M2Y (or M2), PR4, PR5, PR722, B103, SF5, GA-1, and relatedmembers of the Podoviridae family. Specifically, the wildtypebacteriophage Φ29 genome consists of a linear double-stranded DNA(dsDNA) of 19,285 base pairs, having a terminal protein (TP) covalentlylinked to each 5′end. To initiate replication, a histone-like viralprotein forms a nucleoprotein complex with the origins of replicationthat likely contributes to the unwinding of the double helix at both DNAends (Serrano et al., The EMBO Journal 16(9): 2519-2527 (1997)). The DNApolymerase catalyses the addition of the first dAMP to the hydroxylgroup provided by the TP. This protein-primed event occurs opposite tothe second 3′ nucleotide of the template, and the initiation product(TP-dAMP) slides back one position in the DNA to recover the terminalnucleotide. After initiation, the same DNA polymerase replicates one ofthe DNA strands while displacing the other. The high processivity andstrand displacement ability of Φ29 DNA polymerase makes it possible tocomplete replication of the Φ29 TP-containing genome (TP-DNA) in theabsence of any helicase or accessory processivity factors (reviewed bySerrano et al., The EMBO Journal 16(9): 2519-2527 (1997)).

Strand displacement can be enhanced through the use of a variety ofaccessory proteins. They include but are not limited to helicases(Siegel et al., J. BioL Chem. 267:13629-13635 (1992)), herpes simplexviral protein ICP8 (Skaliter and Lehman, Proc. Natl, Acad. Sci. USA91(22):10665-10669 (1994)), single-stranded DNA binding proteins (Riglerand Romano, J. Biol. Chem. 270:8910-8919 (1995)), adenovirus DNA-bindingprotein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164(1994)), and BMRF1 polymerase accessory subunit (Tsurumi et al., J.Virology 67(12):7648-7653 (1993)).

The subject probes can be utilized in an isothermal amplificationreaction. Such amplification reaction does not rely solely upon thermalcycling. The procedure can be applied at a wide range of ambienttemperatures. In particular, denaturation of the double-strandedtemplate sequence is not accomplished solely through an increase intemperature above the melting temperature of the double strandedsequence. Rather, the denaturation process involves physical ormechanical force that separates the strand to allow primer annealing andextension. Various mechanisms for conducting isothermal amplificationreaction including isothermal PCR are described in US. PatentPublication No 20060019274 and U.S. Pat. Nos. 5,824,477 and 6,033,850,which are incorporated herein by reference.

Nucleic acid amplification is generally performed with the use ofamplification reagents. Amplification reagents typically includeenzymes, aqueous buffers, salts, primers, target nucleic acid, andnucleoside triphosphates. Depending upon the context, amplificationreagents can be either a complete or incomplete amplification reactionmixture.

The choice of primers for use in nucleic acid amplification will dependon the target nucleic acid sequence. Primers used in the presentinvention are generally oligonucleotides, e.g., 10 to 100 or 10 to 25bases in length, that can be extended in a template-specific manner viathe action of a polymerase. In general, the following factors areconsidered in primer design: a) each individual primer of a pairpreferably does not self-hybridize in an amplification reaction; b) theindividual pairs preferably do not cross-hybridize in an amplificationreaction; and c) the selected pair must have the appropriate length andsequence homology in order to anneal to two distinct regions flankingthe nucleic acid segment to be amplified. However, not every nucleotideof the primer must anneal to the template for extension to occur. Theprimer sequence need not reflect the exact sequence of the targetnucleic acid. For example, a non-complementary nucleotide fragment maybe attached to the 5′ end of the primer with the remainder of the primersequence being complementary to the target. Alternatively,non-complementary bases can be interspersed into the primer, providedthat the primer sequence has sufficient complementarily with the targetfor annealing to occur and allow synthesis of a complementary nucleicacid strand.

A nucleic acid amplification reaction typically comprises a targetnucleic acid in a buffer compatible with the enzymes used to amplify thetarget. The buffer typically contains nucleotides or nucleotide analogs(ATP, TTP, CTP, GTP, or analogs thereof including without limitationpentaphosphates having the respective base unit) that are capable ofbeing incorporated into a replica strand of the template sequence.

Where desired, amplification reaction is carried out as an automatedprocess. Numerous thermocyclers are available in the art that arecapable of holding 48, 96 or more samples. A suitable optical systemmoves the excitation light from the source to the reaction sites andmeasures the emission light from each sample. For example, multiplefiber optic leads simultaneously read all PCR tubes undergoingthermocycling. However, only a single fluorometer may be needed to readfluorescence from the reaction sites. An analogous detection scheme issuitable in a 96-well microtiter format. This type of format isfrequently desirable in clinical laboratories for large scale samplescreening, for example, for genetic analysis such as screening for AIDSvirus in blood bank screening procedures.

Accordingly, the present invention also provides an apparatus fordetecting the signal generated by the subject probe, which can be usedto detect, measure, and quantify the signal before, during, and afteramplification. The apparatus comprises a thermal unit (e.g., athermocycler) capable of holding an amplification reaction mixturecomprising the subject probes and effecting an amplification of thetarget sequence, and a detector that detects the signal generated fromthe subject probes.

In another embodiment of the present invention, the subject probes areemployed in assays that are conducted on nucleic acid microarrays todetect or quantify nucleic acid targets. In such assays, a fluorescentsignal is generated on a nucleic acid microarray upon the presence of acomplementary target nucleic acid.

Nucleic acid microarrays including gene chips comprise ordered arrays ofnucleic acids that are covalently attached to a solid surface, see e.g.,U.S. Pat. Nos. 5, 871,928, 6,040,193, 6,262,776, 6,403,320, and6,576,424. The fluorescent signal that is generated in the assay can bemonitored and quantified with optical detectors including but notlimited to fluorescence imagers, e.g. commercial instruments supplied byHitachi Corp., San Bruno, Calif. or confocal laser microscopes (confocalfluorescence scanners), e.g. commercial instruments from GeneralScanning, Inc., Watertown, Mass.

In assays that are conducted on nucleic acid microarrays, the targetnucleic acids may be provided as a mixture of nucleic acid sequencesderived from any suitable biological sources. They can be derived frombody fluid, solid tissue samples, tissue cultures or cells derivedtherefrom and the progeny thereof, and sections or smears prepared fromany of these sources, or any other samples that contain nucleic acids.

Where expression pattern is assayed, the mRNA sequences are firsttypically amplified by reverse transcription PCR with universal primersprior to their use as the target sequences in the assay. In oneembodiment, all nucleic acid sequences present in the test sample aresimultaneously applied to the microarray for analysis, thus allowing theinteraction of all target nucleic acid sequences with all nucleic acidsthat are present on the array. In another embodiment, the target nucleicacids applied to the array are pre-selected to yield a subset forrefined hybridization analysis utilizing a microarray. For example, alimited number of target sequences can contain more than one stretch ofspecific nucleotide sequence to be analyzed, e.g. more than one singlenucleotide polymorphism. The nucleic acid sequences of this setting maybe amplified by PCR with the aid of specific primers prior to theiranalysis on the microarray.

In assaying for expression of multiples genes of a subject, targetpolynucleotides are allowed to form stable complexes with probes on theaforementioned arrays in a hybridization reaction. It will beappreciated by one of skill in the art that where antisense RNA is usedas the target nucleic acid, the sequence immobilized on the array arechosen to be complementary to sequences of the antisense nucleic acids.Conversely, where the target nucleic acid pool is a pool of sensenucleic acids, the sequence immobilized on the array are selected to becomplementary to sequences of the sense nucleic acids. Finally, wherethe nucleic acid pool is double stranded, the probes may be of eithersense and/or antisense as the target nucleic acids include both senseand antisense strands.

In one embodiment, labeled probes are utilized to perform a competitivehybridization on a microarray. In this assay format, a target nucleicacid from a test sample competes with a probe of the present inventionfor binding of a known sequence immobilized on the microarray. Theamount of labeled probes that will bind to the immobilized knownsequences is inversely proportional to the concentration ofcorresponding target nucleic acids in the test sample.

A variant hybridization assay involves the use of polymerases on amicroarray to enhance the signals of the probes by performing cleavageof the reporters. For example, a mixture of target sequences are firstallowed to hybridize with known sequences immobilized on the array.Unhybridized sequences are then washed away. Thereafter, probescorresponding to the target sequences are allowed to hybridize todifferent regions on the targets. Upon washing of the excessive unboundprobes, the reporter fluorescent groups on the hybridized probes arecleaved via the action of polymerases, thereby generating a detectablesignal that is indicative of the presence and/or quantity of a targetsequence initially present in the test sample.

Suitable hybridization conditions for use of the labeled probes of theinvention are such that the recognition interaction between the sequenceon the array and target is both sufficiently specific and sufficientlystable. As noted above, hybridization reactions can be performed underconditions of different “stringency”. Relevant conditions includetemperature, ionic strength, time of incubation, the presence ofadditional solutes in the reaction mixture such as formamide, and thewashing procedure. Higher stringency conditions are those conditions,such as higher temperature and lower sodium ion concentration, whichrequire higher minimum complementarity between hybridizing elements fora stable hybridization complex to form. Conditions that increase thestringency of a hybridization reaction are widely known and published inthe art. See, for example, (Sambrook, et al., (1989), supra).

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. In a preferred embodiment, washingthe hybridized array prior to detecting the target-probe complexes isperformed to enhance the signal to noise ratio. Typically, thehybridized array is washed at successively higher stringency solutionsand signals are read between each wash. Analysis of the data sets thusproduced will reveal a wash stringency above which the hybridizationpattern is not appreciably altered and which provides adequate signalfor the particular polynucleotide probes of interest. Parametersgoverning the wash stringency are generally the same as those ofhybridization stringency. Other measures such as inclusion of blockingreagents (e.g. sperm DNA, detergent or other organic or inorganicsubstances) during hybridization can also reduce non-specific binding.

Imaging specific hybridization event on a microarray is typicallyperformed with the aid of an optical system. Non-limiting examples ofsuitable systems include camera, charge couple device (CCD),electron-multiplying charge-coupled device (EMCCD), intensified chargecoupled device (ICCD), and confocal microscope.

The microarray provides a positional localization of the sequence wherehybridization has taken place. The position of the hybridized regioncorrelates to the specific sequence, and hence the identity of thetarget expressed in the test sample. The detection methods also yieldquantitative measurement of the level of hybridization intensity at eachhybridized region, and thus a direct measurement of the level ofexpression of a given gene transcript. A collection of the dataindicating the regions of hybridization present on an array and theirrespective intensities constitutes a hybridization pattern that isrepresentative of a multiplicity of expressed gene transcripts of asubject. Any discrepancies detected in the hybridization patternsgenerated by hybridizing target polynucleotides derived from differentsubjects are indicative of differential expression of a multiplicity ofgene transcripts of these subjects.

In one aspect, the hybridization patterns to be compared can begenerated on the same array. In such case, different patterns aredistinguished by the distinct types of detectable labels. In a separateaspect, the hybridization patterns employed for the comparison aregenerated on different arrays, where discrepancies are indicative of adifferential expression of a particular gene in the subjects beingcompared.

The test nucleic acids for a comparative hybridization analysis can bederived from (a) cells from different organisms of the same species(e.g. cells derived from different humans); (b) cells derived from thesame organism but from different tissue types including normal ordisease tissues, embryonic or adult tissues; (c) cells at differentpoints in the cell-cycle; (d) cells treated with or without external orinternal stimuli. Thus, the comparative hybridization analysis using thearrays of the present invention can be employed to monitor geneexpression in a wide variety of contexts. Such analysis may be extendedto detecting differential expression of genes between diseased andnormal tissues, among different types of tissues and cells, amongstcells at different cell-cycle points or at different developmentalstages, and amongst cells that are subjected to various environmentalstimuli or lead drugs. Therefore, the expression detecting methods ofthis invention may be used in a wide variety of circumstances includingdetection of disease, identification and quantification of differentialgene expression between at least two samples, linking the differentiallyexpressed genes to a specific chromosomal location, and/or screening forcompositions that upregulate or downregulate the expression or alter thepattern of expression of particular genes.

The subject amplification and any other hybridization assays describedherein can be used to detect any target nucleic acids from any sourcessuspected to contain the target. It is not intended to be limited asregards to the source of the sample or the manner in which it is made.Generally, the test sample can be biological and/or environmentalsamples. Biological samples may be derived from human or other animals,body fluid, solid tissue samples, tissue cultures or cells derivedtherefrom and the progeny thereof, sections or smears prepared from anyof these sources, or any other samples that contain nucleic acids.Preferred biological samples are body fluids including but not limitedto urine, blood, cerebrospinal fluid, spinal fluid, sinovial fluid,semen, ammoniac fluid, cerebrospinal fluid (CSF), and saliva. Othertypes of biological sample may include food products and ingredientssuch as dairy items, vegetables, meat and meat by-products, and waste.Environmental samples are derived from environmental material includingbut not limited to soil, water, sewage, cosmetic, agricultural andindustrial samples, as well as samples obtained from food and dairyprocessing instruments, apparatus, equipment, disposable, andnon-disposable items.

Polynucleotides labeled according to the invention may also be used ingel shift assays. Such an assay, also known as electrophoretic mobilityshift assay (EMSA), gel mobility shift assay, band shift assay, or gelretardation assay, is a common technique used to study protein-DNA orprotein-RNA interactions. This procedure can determine if a protein ormixture of proteins is capable of binding to a given DNA or RNAsequence, and can sometimes indicate if more than one protein moleculeis involved in the binding complex. Labeled oligonucleotides may be usedin gel shift assays by performing electrophoresis and subsequentlydetermining the extent of migration of the labeled oligonucleotides inthe gel by visualizing the emission of the fluorescent label. Gel shiftassays may be performed in vitro concurrently with DNase footprinting,primer extension, and promoter-probe experiments when studyingtranscription initiation, DNA replication, DNA repair or RNA processingand maturation. Methods of performing gel shift assays are known. See,e.g. Garner, M. M. and Revzin, A. (1981) “A gel electrophoresis methodfor quantifying the binding of proteins to specific DNA regions:application to components of the Escherichia coli lactose operonregulatory system.” Nucleic Acids Res. 9:3047-3060 or Fried, M. andCrothers, D. M. (1981) “Equilibria and kinetics of lacrepressor-operator interactions by polyacrylamide gel electrophoresis.”Nucleic Acids Res., 9:6505-6525.

Fluorescently labeled polypeptides of the invention are useful in a widevariety of assays. Such assays can be performed to discern specificprotein-protein interactions, protein-nucleic acid interaction,interactions between a protein of interest and candidate inhibitors oractivators. Candidate inhibitors or activators include but are notlimited to antisense oligonucleotides, double stranded RNAs, ribozymes,a ribozyme derivatives, antibodies, liposomes, small molecules,inorganic or organic compounds. The subject assays can also be performedto study enzymatic kinetics, for e.g., drug design, screen and/oroptimization and can be performed using the fluorescently labeledpolypeptides in solution or immobilized on a solid substrate.

Of particular interest is a specific interaction between a cell surfacereceptor and its corresponding ligand. Cell surface receptors aremolecules anchored on or inserted into the cell plasma membrane. Theyconstitute a large family of proteins, glycoproteins, polysaccharidesand lipids, which serve not only as structural constituents of theplasma membrane, but also as regulatory elements governing a variety ofbiological functions. In another aspect, the specific protein-proteininteraction involves a cell surface receptor and an immunoliposome or animmunotoxin. In yet another aspect, the specific protein-proteininteraction may involve a cytosolic protein, a nuclear protein, achaperone protein, or proteins anchored on other intracellularmembranous structures. In yet another aspect, the specificprotein-protein interaction is between a target protein (e.g., anantigen) and an antibody specific for that antigen.

A specific interaction between a labeled polypeptide and an interactingentity is assayed by mixing the two entities under conditions suchinteraction is suspected to occur. Typically, the interaction isvisualized with the aid of an optical device. Where desired, theseentities can be placed within an optical confinement (see, e.g., U.S.Pat. Nos. 7,267,673, and 7,170,050). Where single molecule is to bedetected, each optical confinement contains only one target that isbeing investigated. This can be achieved by diluting a minute amount oftarget in a large volume of solution, such that deposition over an arrayof confinements results in a primary distribution, or a majority ofconfinements will have a single target molecule disposed there. Thelabeled polypeptide and the interacting entity can be immobilized ontothe inner surface of the optical confinement by any of the methodsavailable in the art. Such methods encompass the uses of covalent andnoncovalent attachments effected by a variety of binding moieties. Thechoice of the binding moieties will depend on the nature of the labeledpolypeptide and/or the interacting entity. One way to immobilize thelabeled polypeptide or the proteinaceous probe involves the use of thestreptavidin or avidin/biotin binding pair.

In one embodiment, the polypeptide to be reacted with a compound of theinvention comprises 3 to about 80 amino acids. Examples of suchpolypeptides include, but are not limited to, neuropeptides, cytokines,toxins and peptidase or protease substrates. Fluorescentlylabeled-neuropeptides, -cytokines and -toxins may be used to map orvisualize the distribution of the receptors specific to the respectivepeptides. As an example, when labeled with a compound of the invention,phalloidin, which is a toxin with a cyclic peptide structure, can beused to stain F-actin filaments in cells. As another example, whenlabeled with a fluorescent group of the invention, α-bungarotoxin, apeptide-based snake toxin, can be used to detect acetylcholine receptor.Peptidase or protease substrates labeled with a fluorescent group of theinvention may be used to assay the activities of the peptidases orproteases, and used in screening drugs designed as inhibitors of thepeptidases or proteases. For example, a peptide comprising a peptidesequence cleavable by a peptidase may be labeled at one end of thepeptide sequence with a first fluorescent group, a fluorescence donorfluorescent group, selected from a fluorescent group of the inventionand at the other end of the peptide sequence with a second fluorescentgroup, a fluorescence acceptor fluorescent group (such as anotherfluorescent group from the invention or a quencher), where the first dyeand second dye form a fluorescence resonance energy transfer (FRET)pair. By detecting the fluorescence difference of either the donorfluorescent group or the acceptor fluorescent group of the FRET pairbefore and after the peptide is cleaved by said peptidase, the level ofenzyme activity can be assessed.

Other polypeptide conjugates that can be prepared according to theinvention include those of antibodies, lectins, enzymes, lipoproteins,albumins, avidin, streptavidin, annexins, protein A, protein G,transferrin, apotransferrin, phycobiliproteins and other fluorescentproteins, toxins, growth factors, tubulins, hormones, various receptorsand ion channels.

In one embodiment, compounds of the invention may be reacted withantibodies. Such antibodies may be primary or secondary depending on thedesired application. If the antigen to be detected is present in verysmall amounts, a secondary antibody may be used in order to providesignal amplification. Various secondary antibody isotypes may belabeled. Non-limiting examples of secondary antibody isotypes areAnti-mouse IgG, Anti-mouse IgM, Anti-rabbit IgG, Anti-rat IgG, Anti-ratIgM, Anti-guinea pig IgG, Anti-chicken IgG, Anti-hamster IgG, Anti-humanIgG, Anti-human IgM, Anti-goat IgG, Anti-mouse IgG, Anti-rabbit IgG,Anti-rat IgG, Anti-sheep IgG, Anti-goat IgG, Anti-mouse IgG, Anti-humanIgG, Anti-rat IgG, Anti-mouse IgG , Anti-human IgG, Anti-rat IgG,Anti-goat IgG, and Anti-rabbit IgG.

Alternatively, Fab fragments may be labeled with the compounds of theinvention. Such fragments may be superior to whole antibody conjugatesbecause they lack the Fc region, which would reduce nonspecificinteractions with Fc receptor-bearing cell membranes and would allowbetter penetration into tissues.

Labeled secondary antibodies of the invention may be used in signalamplification kits such as those commercialized by Molecular Probes,Inc. Such kits could each provide two labeled antibodies specific to aprimary antibodies, such as a mouse antibody. In one embodiment, arabbit anti-mouse IgG antibody conjugate of the invention is first usedto bind to the mouse-derived primary antibody. The fluorescence is thendramatically enhanced by the addition of a second conjugate of a goatanti-rabbit IgG antibody.

In yet another embodiment, the compounds of the invention may be used tolabel protein A and/or protein G. Protein A and protein G are bacterialproteins that bind with high affinity to the Fc portion of variousclasses and subclasses of immunoglobulins from a variety of species,such as Bovine, Cat, Chicken, Dog, Goat, Guinea pig, Horse, Human IgG1,IgG2, IgG3, IgG4, Human IgM, IgA, IgE, Human IgD, Mouse IgG1 or others,Pig, Rabbit, Rat or Sheep, which may be used in the detection ofimmunoglobulins. Alternatively, immunoglobins can be labeled with acompound of the invention having a structure of Formula I and retainsbinding specificity to its target after such labeling. These labeledimmunoglobins can be used for in-vitro or in-vivo detection of thetarget antigen. In some embodiments, the labeled immunoglobins comprisea fluorophore that has an absorption maximal wavelength equal to 405±4nm. In other embodiments labeled immunoglobins comprise a fluorophorethat has an absorption maximal wavelength about 470±4 nm. In variousembodiments of the invention, such labeled immunoglobins bind to anantigen on a cancer cell. In some embodiments, the labeled immunoglobinbinds to erb2.

Labeled antibodies prepared according to the invention may be primaryantibodies for various applications. While secondary detection methodscan provide significant signal amplification, a directly labeled primaryantibody often produces lower background fluorescence and lessnonspecific binding. Using primary antibodies also allows multipleprimary antibodies of the same isotype or derived from the same speciesto be used in the same experiment when they are directly labeled.

Examples of such primary antibodies include polyclonal antibodiesspecific for reporter gene products. These includeAnti-Green-Fluorescent Protein Antibodies, Anti-GlutathioneS-Transferase Antibody, Anti-beta-Glucuronidase Antibody,Anti-beta-Galactosidase Antibody, Monoclonal Antibodies Specific forEpitope Tags, Penta-His Antibody, Anti-HA Antibody and Anti-c-mycAntibody.

Organelle-specific labeled antibodies may also be prepared to labelvarious subcellular organelles and components such as the endoplasmicreticulum, peroxisomes, mitochondria, or cytochrome c. Labeledantibodies may also be specific for proteins in the oxidativephosphorylation system, such as antibodies against cytochrome oxidase(Complex IV) or antibodies against Complexes I, II, III and V, or othermitochondrial proteins such as anti-mitochondrial porin antibodies oranti-pyruvate dehydrogenase antibodies.

In other embodiments, labeled antibodies specific for proliferationmarkers and cell-cycle control proteins may be prepared. Such antibodiesinclude Anti-Bromodeoxyuridine Antibody (Anti-BrdU Antibody), which mayfor example be used in TUNEL assays, Anti-Human mRNA-Binding Protein HuRAntibody (Anti-HuR Antibody), Anti-Human Neuronal Protein HuC/HuDAntibody (Anti-Hu Antibody), Anti-cdc6 Peptide Antibody, Anti-CDAntibodies, Antibodies against D Cyclins/Cyclin-Dependent KinaseInhibitors, and Anti-Phosphoinositide Antibodies.

Some labeled antibodies may be specific for structural cellularproteins. Examples of such antibodies are Anti-alpha-Tubulin MonoclonalAntibody, Anti-Glial Fibrillary Acidic Protein (GFAP) Antibody,Anti-Desmin Antibody, or Anti-Fibronectin Antibody. Additionalantibodies suitable for use in the invention include antibodies specificfor neuronal proteins such as Anti-Synapsin I Antibody or Anti-NMDAReceptor Antibodies. Other Polyclonal and Monoclonal Antibodies that maybe labeled according to the invention include Anti-Human Golgin-97Antibody, Anti-Human Transferrin Receptor Antibody, Antibodies againstMatrix Metalloproteinases and Anti-Bovine Serum Albumin Antibody.

The specific interaction between an antigen and an antibody has beenexplored in the context of immunoassays utilizing the subjectfluorescent compounds. The immunoassays can permit single-moleculedetection or ensemble detection. The subject immunoassays can beperformed to characterize biological entities, screen for antibodytherapeutics, and determine the structural conformations of a targetantigen. For instance, immunoassays involving antibodies that arespecific for the biological entity or specific for a by-product producedby the biological entity have been routinely used to identify the entityby forming an antibody-entity complex. Immunoassays are also employed toscreen for antibodies capable of activating or down-regulating thebiological activity of a target antigen of therapeutic potential.Immunoassays are also useful for determining structural conformations byusing anti-idotypic antibodies capable of differentiating targetproteins folded in different conformations.

According to one embodiment of the invention, biomolecules labeled witha fluorescent group of the invention such as proteins are suitable forin vivo imaging, including without limitation imaging a biomoleculepresent inside a cell, a cell, tissue, organ or a whole subject. Wheredesired, the labeled biomolecules can be used to perform “In CellWestern” in which given molecules (e.g., a specific cellular protein)present inside a cell are stained and imaged.

The compounds of the invention may also be used to produce labeledbiomolecules for use in immunohistochemistry and immunocytochemistryexperiments. In immunohistochemistry (IHC), the presence and location ofproteins is determined within a tissue section by exploiting theprinciple of an antibody binding specifically to an antigens present ina biological tissue. Such experiments may, for example, be used in thediagnosis and treatment of cancer. Specific molecular markers arecharacteristic of particular cancer types and are known to personsskilled in the art. IHC can also, be used in basic research to determinethe distribution and localization of biomarkers in different parts of atissue. Visualization of antibody-antigen interactions can beaccomplished by reacting an antibody with a reactive fluorescentcompound of the invention and using the labeled antibody to stain tissuesections. In immunocytochemistry, the labeled antibody is used to stainpopulations of cultured cells. These techniques can be combined withconfocal laser scanning microscopy, which is highly sensitive and canalso be used to visualise interactions between multiple proteins.Subcellular localization of proteins may also be possible using confocalmicroscopy.

Of particular interest is the use of the labeled polypeptide forconducing immunocytochemistry. Fluorescence immunocytochemistry combinedwith fluorescence microscopy provides visualization of biomolecules suchas proteins and nucleic acids within a cell. One method uses primaryantibodies hybridized to the desired target. Then, secondary antibodiesconjugated with the subject fluorescent dyes and targeted to the primaryantibodies are used to tag the complex. The complex is visualized byexciting the dyes with a wavelength of light matched to the dye'sexcitation spectrum.

Immunocytochemistry can also be employed to discern subcellularlocalization of a given protein or nucleic acid. For instance,colocalization of biomolecules in a cell is performed using differentsets of antibodies for each cellular target. For example, one cellularcomponent can be targeted with a mouse monoclonal antibody and anothercomponent with a rabbit polyclonal antibody. These are designated as theprimary antibody. Subsequently, secondary antibodies to the mouseantibody or the rabbit antibody, conjugated to different fluorescentdyes of the present invention having different emission wavelengths, areused to visualize the cellular target.

The compounds of the invention or the labeled biomolecules of theinvention can also be used to label cells or particles for a variety ofapplications. Accordingly, the present invention provides a method ofindividually labeling a cell within a population of cells whereby thecell is differentially labeled relative to neighboring cells within thepopulation. The method typically comprises contacting the cell with alabeled biomolecule of the present invention, wherein said biomoleculecomprises a targeting moiety that binds to a binding partner that isindicative of said cell, and thereby differentially labeling the cellrelative to neighboring cells within the population. The targetingmoiety can be any biomolecules that recognize a binding partner on thecell to be detected. The choice of the targeting moiety will varydepending on the cell that is to be labeled. For example, for detectinga cancer cell, a targeting moiety is selected such that its bindingpartner is differentially expressed on a cancer cell. A vast number ofcancer markers are known in the art. They include without limitationcell surface receptors such as erb2, PDGF receptor, VEGF receptors, ahost of intracellular proteins such as phosphatidylinositol 3-kinases,c-abl, raf, ras, as well as a host of nuclear proteins includingtranscription factors and other nucleic acid binding molecules. In someother embodiments, the cancer marker is Immunoglobulin epsilon Fcreceptor II, Alk-1,CD20, EGF receptor, FGF receptor, NGF receptor,EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55,CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin,Mesothelin receptor, Nogo receptor, folate receptor, CXCR4, insulin-likegrowth factor receptor, Ganglioside GD3, and alpha or beta Integrins. Todifferentially label various cell types, targeting moieties recognizinga cell-specific binding partner can be used. For example, there are ahost of protein markers differentially expressed on T cells as opposedon B cells or other cells of different lineage. Neuronal markers, musclecell markers, as well as markers indicative of cells of ectodermal,mesodermal or endodermal origins are also known in the art, all of whichcan be used depending on the intended applications. The targetingmoieties can be antibodies, receptors, cytokines, growth factors, andany other moieties or combinations thereof that are recognized by abinding partner on the cell to be labeled. The cell which is labeled maybe labeled intracellularly.

The differentially labeled cells can be imaged by directing excitingwavelength to the cell and detecting emitted fluorescence from the cell,in a number of in-vitro formats, either in solution or immobilized on asubstrate.

The labeled cells and/or the intensity of the fluorescence may bedetected or quantified by performing flow cytometry. Cells or particleslabeled with the compounds of the invention or stained with labeledbiomolecules of the invention may also be separated and isolated basedon the specific properties of the label using fluorescence activatedcell sorting (FACS). Such techniques are known in the art. Briefly,cells are labeled with a subject fluorescent dye and then passed, in asuspending medium, through a narrow dropping nozzle so that each cell istypically in a small droplet. A laser based detector system is used toexcite fluorescence and droplets with positively fluorescent cells aregiven an electric charge. Charged and uncharged droplets are separatedas they fall between charged plates and so collect in different tubes.The machine can be used either as an analytical tool, counting thenumber of labeled cells in a population or to separate the cells forsubsequent growth of the selected population. Further sophistication canbe built into the system by using a second laser system at right anglesto the first to look at a second fluorescent label or to gauge cell sizeon the basis of light scatter.

Additional guidance for performing fluorescent cell sorting can be foundin publications such as the following: Darzynkiewicz, Z., Crissman, H.A. and Robinson, J. P., Eds., Cytometry, Third Edition Parts A and B(Methods in Cell Biology, Volumes 63 and 64), Academic Press (2001);Davey, H. M. and Kell, D. B., “Flow cytometry and cell sorting ofheterogeneous microbial populations: the importance of single-cellanalyses,” Microbiological Rev 60, 641-696 (1996); Givan, A. L., FlowCytometry: First Principles, Second Edition, John Wiley and Sons (2001);Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O., Roederer, M. andHerzenberg, L. A., “The history and future of the fluorescence activatedcell sorter and flow cytometry: a view from Stanford,” Clin Chem 48,1819-1827 (2002); Jaroszeski, M. J. and Heller, R., Eds., Flow CytometryProtocols (Methods in Molecular Biology, Volume 91), Humana Press(1997); Ormerod, M. G., Ed., Flow Cytometry: A Practical Approach, ThirdEdition, Oxford University Press (2000); Robinson, J. P., Ed., CurrentProtocols in Cytometry, John Wiley and Sons (1997); Shapiro, H. M.,“Optical measurement in cytometry: light scattering, extinction,absorption and fluorescence,” Meth Cell Biol 63, 107-129 (2001);Shapiro, H. M., Practical Flow Cytometry, Fourth Edition, Wiley-Liss(2003); Weaver, J. L., “Introduction to flow cytometry,” Methods 21,199-201 (2000).

Fluorescent compounds of the invention may also be used for fluorescencelifetime imaging (FLIM). FLIM is a useful technique for producing imagesbased on the variation in the fluorescence decay characteristics of afluorescent sample. It can be used as an imaging technique in confocalmicroscopy and other microscope systems. The lifetime of the fluorophoresignal, rather than its intensity, is used to create the image in FLIM,which has the advantage of minimizing the effect of photon scattering inthick layers of sample. FLIM may be useful for biomedical tissueimaging, allowing to probe greater tissue depths than conventionalfluorescence microscopy.

The compounds of the invention may be used in single moleculeapplications. Removal of ensemble averaging by observing individualmolecules of fluorescent group may allow the determination of themechanism of biological and chemical processes. Such processes mayinclude the translocation of protein motors such as kinesin or myosin,formation, dissolution and translocation of cellular protein complexesand the mechanism of action of DNA or RNA polymerases. In suchexperiments, the present compounds may be used, for example, to labelbiomolecules which are attached to a surface such as a microscopy slideor flow chamber. Individual fluorophores may subsequently be observedusing total internal reflection fluorescence microscopy.

The present compounds may also be used for the labeling of lipids.Lipids are involved in many biological processes, and the labeling oflipids and lipid rafts may is often a valuable method for studying theirproperties. Various lipid monolayers and bilayers may be labeled in livecells or artificial systems such as liposomes and micelles. For example,a live cell population may be labeled with a fluorescent conjugateprepared by reacting a compound of the invention and cholera toxinsubunit B, which specifically interacts with lipid rafts. Such lipidrafts may then be crosslinked into distinct membrane patches by the useof an anti-cholera toxin antibody, which may be labeled with one of thepresent compounds.

The labeled polypeptides of the present invention find use as biosensorsin prokaryotic and eukaryotic cells, e.g. as calcium ion indicators, aspH indicators, as phosphorylation indicators, as indicators of otherions including without limiting to magnesium, sodium, potassium,chloride and halides. For example, for detection of calcium ion,proteins containing an EF-hand motif are known to translocate from thecytosol to membranes upon binding to calcium ion. These proteins containa myristoyl group that is buried within the molecule by hydrophobicinteractions with other regions of the protein. Binding of calcium ioninduces a conformational change exposing the myristoyl group which thenis available for the insertion into the lipid bilayer. Labeling such anEF-hand containing protein with a subject fluorescent dye makes it anindicator of intracellular calcium ion concentration by monitoring thetranslocation from the cytosol to the plasma membrane. Such monitoringcan be performed with the use of an optical detector, e.g., a confocalmicroscope. EF-hand proteins suitable for use in this system include,but are not limited to: recoverin (1-3), calcineurin B, troponin C,visinin, neurocalcin, calmodulin, parvalbumin, and the like.

For use as a pH indicator, a system based on hisactophilins may beemployed. Hisactophilins are myristoylated histidine-rich proteins knownto exist in Dictyostelium. Their binding to actin and acidic lipids issharply pH-dependent within the range of cytoplasmic pH variations. Inliving cells membrane binding seems to override the interaction ofhisactophilins with actin filaments. At pH of approximately 6.5 theytypically locate to the plasma membrane and nucleus. In contrast, at pH7.5 they evenly distribute throughout the cytoplasmic space. This changeof distribution is reversible and is attributed to histidine clustersexposed in loops on the surface of the molecule. The reversion ofintracellular distribution in the range of cytoplasmic pH variations isin accord with a pK of 6.5 of histidine residues. The cellulardistribution is independent of myristoylation of the protein. Byconjugating the subject fluorescent dye to hisactophilin, theintracellular distribution of the labeled hisactophilin can be followedby laser scanning, confocal microscopy or standard fluorescencemicroscopy. Quantitative fluorescence analysis can be done by performingline scans through cells (laser scanning confocal microscopy) or otherelectronic data analysis (e.g., using metamorph software (UniversalImaging Corp) and averaging of data collected in a population of cells.

The subject fluorescent proteins also find use in applications involvingthe automated screening of arrays of cells by using microscopic imagingand electronic analysis. Screening can be used for drug discovery and inthe field of functional genomics: e.g., where the subject proteins areused as markers of whole cells to detect changes in multicellularreorganization and migration, e.g., formation of multicellular tubules(blood vessel formation) by endothelial cells, migration of cellsthrough Fluoroblok Insert System (Becton Dickinson Co.), wound healing,neurite outgrowth; where the proteins are used as markers fused topeptides (e.g., targeting sequences) and proteins that allow thedetection of change of intracellular location as indicator for cellularactivity, for example: signal transduction, such as kinase andtranscription factor translocation upon stimuli, such as protein kinaseC, protein kinase A, transcription factor NFkB, and NFAT; cell cycleproteins, such as cyclin A, cyclin B1 and cyclinE; protease cleavagewith subsequent movement of cleaved substrate, phospholipids, withmarkers for intracellular structures such as endoplasmic reticulum,Golgi apparatus, mitochondria, peroxisomes, nucleus, nucleoli, plasmamembrane, histones, endosomes, lysosomes, microtubules, actin.

The examples below are for the purpose of illustrating the practice ofthe invention. They shall not be construed as being a limitation on thescope of the invention or claims.

EXAMPLES Example 1 Preparation of8-(5-carboxypentoxy)pyrene-1,3,6-trisulfonic acid

A mixture of 8-hydroxypyrene-1,3,6-trisulfonate sodium salt (2.6 g),diisopropylethylamine (2 g) and ethyl 6-bromohexanoate (7 g) in methanol(150 mL) was refluxed for 24 hours. The solvent was removed by rotaryevaporation. The product was purified by HPLC. The isolated ethyl esterwas redissolved in methanol (100 mL). About 0.5 g NaOH dissolved in 25mL water was added. The resulting solution was stirred overnight. Themethanol was removed by rotary evaporation. To the remaining aqueoussolution was added acid until the solution became acidic. Theprecipitate was collected by filtration and dried under vacuum to give8-(5-carboxypentoxy)pyrene-1,3,6-trisulfonic acid.

Example 2 Preparation of Compound 1

To a solution of 8-(5-carboxypentoxy)pyrene-1,3,6-trisulfonic acid (150mg) in 5 mL DMF were added 5 equivalents of triethylamine and 1equivalent of TSTU (O-succinimidyl-N,N,N,N′-tetramethyluroniumtetrafluoroborate). The resulting solution was stirred for 2 hours. Thesolution was concentrated to about 1 mL and then mixed with 10 mL CH₃CN.The resulting precipitate was collected and dried under vacuum.

Example 3 Preparation of the Free Acid of Compound 2

To a solution of compound 1 (50 mg) in DMF (5 mL) were addedamino-PEO12-propionic acid (1.2 equivalent) and triethylamine (1.2equivalent). The resulting solution was stirred at room temperature for24 hours. The solvent was removed under high vacuum at room temperature.The product was purified by LH-20 Sephadex column eluting with water.

Example 4 Preparation of Compound 2

Compound 2 was prepared according to the procedure described above inExample 2.

Example 5 Preparation of 1-(5-ethoxycarbonylpentyl)aminopyrene

A mixture of 1-aminopyrene (5 g), potassium carbonate (15 g) and ethyl6-bromohexanoate (10 g) in 60 mL acetonitrile was refluxed for 24 hours.The solvent was removed and the product was purified by a silica gelcolumn eluting with EtOAc/hexane.

Example 6 Preparation of 8-(carboxypentyl)aminopyrene-1,3,6-trisulfonicacid

1-(5-ethoxycarbonylpentyl)aminopyrene (1 g) was added in small portionto a stirred solution of 30% fuming sulfuric acid (8 mL) cooled to −30°C. After the addition, the solution was slowly warmed to roomtemperature and stirring continued at room temperature for 5 hours. Thereaction mixture was poured over ice and the resulting precipitate wascollected by suction filtration. The crude product was purified by HPLCon a C18 reverse phase column using triethylammonium acetate as theeluting buffer. The collected fractions containing the desired productwere pooled and concentrated. The concentrate was acidified using HCl toprecipitate out the product, which was then dried on a lyophilizer.

Example 7 Preparation of Compound 3

Compound 3 was prepared from8-(carboxypentyl)aminopyrene-1,3,6-trisulfonic acid (Example 6)according to the procedure described in Example 2.

Example 8 Preparation of 1-(N-phthalimidobutyl)aminopyrene

A mixture of 1-aminopyrene (5 g), potassium carbonate (15 g) andN-(4-bromobutyl)phthalimide (15 g) in 150 mL CH₃CN was refluxed for 24hours. The solvent was removed by rotary evaporation and the crudeproduct was purified by a silica gel column eluting with EtOAc/hexane.

Example 9 Preparation of Compound 4

1-(N-Phthalimidobutyl)aminopyrene (1 g) was added in small portion to astirred solution of 30% fuming sulfuric acid (8 mL) cooled to −30° C.After the addition, the solution was slowly warmed to room temperatureand stirring continued at room temperature for 5 hours. The reactionmixture was poured over ice and the resulting precipitate was collectedby suction filtration. The sulfonated intermediate was dissolved inethanol (50 mL) and then treated with ˜5 equivalents of hydrazine atreflux temperature for 8 hours. The mixture was cooled to roomtemperature, followed by filtration to remove the precipitate. Thefiltrate was concentrated down to give the crude product, which was thenpurified by HPLC. The isolated product was converted to the potassiumsalt using Dowex potassium resin to exchange the cations.

Example 10 Preparation of Compound 5

Compound 4 (500 mg) in 20 mL anhydrous DMF was treated with2-(N-t-Boc-aminooxy)acetic acid succinimidyl ester (Biotium) for 24hours. The solvent was removed under high vacuum at room temperature.The residue was triturated with CH₃CN (5 mL) and then filtered and driedunder vacuum. The solid was added in small portions to a stirredsolution of 50% trifluoroacetic acid in CH₂Cl₂ (50 mL) cooled in an icebath. The mixture was stirred for ˜8 hours. The solvent was removed andthe residue was subject to HPLC to give pure product compound 5 as atriethylammonium salt, which was further treated with potassium Dowexresin to give the product as a potassium salt.

Example 11 Preparation of Protein Dye-Conjugates

Fluorescent conjugates of goat anti-mouse IgG (GAM) and streptavidinwere prepared from the respective proteins and a reactive dye, followingpublished procedures (U.S. Pat. No. 6,974,873; Haugland et al., Meth.Mol. Biol. 45, 205(1995); Haugland et al., Meth. Mol. Biol. 45,223(1995); Haugland et al., Meth. Mol. Biol. 45, 235(1995); Haugland etal., Current Protocols in Cell Biology, 16.5.1-16.5.22(2000)). Briefly,an antibody or streptavidin at 1 mg/mL in 0.1 mM pH 8.5 sodiumbicarbonate buffer was mixed with one of the reactive dye at variousratio of dye molecules/protein molecule. After incubating for about anhour at room temperature, the reaction mixture was separated by gelfiltration using Sephadex G-25 equilibrated with PBS (pH 7.4). Thevarious dye molecules/protein ratios used in the labeling reactionsproduced protein conjugates with different degree of dye labeling (DOL)as listed in Table 3 below for each dye/protein pair.

TABLE 3 List of selected antibody and streptavidin conjugates preparedaccording to the invention Degree of Labeling Protein Dye (DOL)Streptavidin Compound 1 Goat anti-mouse IgG Alexa Fluor ® 405 SE 1.5;4.0; 5.4; 6.4 Goat anti-mouse IgG Compound 2 1.6; 4.8; 7.2; 9.5

Example 12 Flow Cytometry Analysis of Cells Intracellularly Stained withDye-Antibody Conjugates

One million Jurkat cells were fixed, permeabilized, and incubated with0.25 μg mouse anti-human CD3 antibody (BD Biosciences). The CD3 antibodywas followed by incubation with 1 μg goat anti-mouse IgG labeled withcompound 2 or Alexa Fluor® 405 at an indicated DOL. About 10,000 cellsfrom each sample were analyzed on a BD LSRII flow cytometer andfluorescence was detected in the channel fitted with a 450/50 bandfilter. To compare the background staining of the labeled secondaryantibodies, the staining experiments were also carried out with theprimary antibody being replaced with an isotype primary antibody. Thedata is presented in FIG. 3.

Example 13 Intracellular Staining of Jurkat Cells with Goat Anti-MouseIgG Labeled with Compound 2

HeLa cells were fixed, permeabilized and stained with mousealpha-tubulin antibody followed by 5 ug/mL goat anti-mouse IgG labeledwith compound 2. Images were captured on an Olympus mercury arc lampmicroscope at 60× using a CCD camera and ImagePro Express software.

1. A compound of Formula I:

wherein: A is —O— or where R₂ is —H or alkyl; each R₁ and R₁′ isindependently —H or alkyl, where each alkyl may be the same or differentand where any pair of two alkyl groups may combine to form a cyclicring; a is an integer between 2 and 20; L is a bond or a covalent linkercomprising between 1 and 100 atoms; and R_(x) is a reactive groupcapable of forming a covalent bond upon reacting with a reactionsubstrate.
 2. The compound of claim 1, wherein A is —O—.
 3. The compoundof claim 1, wherein A is —NR₂—.
 4. The compound of claim 1, wherein A is—NH—.
 5. The compound of claim 1, wherein a is between 2 and
 10. 6. Thecompound of claim 1, wherein a is
 5. 7. The compound of claim 1, whereineach R₁ and R₁′ is H.
 8. The compound of claim 1, wherein L is a bond.9. The compound of claim 1, wherein L comprises a water-soluble moiety.10. The compound of claim 1, wherein R_(x) is a reactive group capableof forming a covalent bond by reacting with an amine group.
 11. Thecompound of claim 1, wherein R_(x) is an activated ester.
 12. Thecompound of claim 1, wherein R_(x) has the formula —CO-(Lg), wherein Lgis a leaving group.
 13. The compound of claim 1, wherein R_(x) is anN-hydroxysuccinimide ester.
 14. The compound of claim 1, wherein R_(x)is an aminooxy.
 15. The compound of claim 4, wherein R_(x) is anaminooxy.
 16. A kit comprising: i) the compound of claim 1; ii) abuffer; iii) materials or devices for purifying conjugation products;and iv) instructions instructing the use of the compound.
 17. Abiomolecule comprising a label derived from a compound of Formula I,wherein the at least one reactive moiety of Formula I has undergone areaction which attaches the label to the biomolecule.
 18. Thebiomolecule comprising a label of claim 17 wherein the biomoleculecomprises a polynucleotide.
 19. The biomolecule comprising a label ofclaim 17 wherein the biomolecule comprises a polypeptide.
 20. Thebiomolecule of claim 19, wherein the polypeptide further comprises anantigen binding site.
 21. The biomolecule of claim 19, wherein thepolypeptide is a whole immunoglobulin.
 22. The biomolecule of claim 19,wherein the polypeptide is a Fab fragment.
 23. An immunoglobincomprising a label derived from a compound of Formula I, wherein the atleast one reactive moiety of Formula I has undergone a reaction whichattaches the label to the immunoglobin, wherein the immunoglobin is anantibody that binds specifically to an antigen on a cancer cell.
 24. Theimmunoglobulin of claim 23, wherein the antibody binds to erb2.
 25. Amethod of preparing a labeled biomolecule comprising reacting a compoundaccording to claim 1 and a substrate biomolecule under conditionssufficient to effect crosslinking between the compound and the substratebiomolecule.
 26. The method of claim 25, wherein the substratebiomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipidor a combination thereof.
 27. The method of claim 25, wherein thesubstrate biomolecule is a polynucleotide.
 28. A method for labeling acell within a population of cells whereby the cell is differentiallylabeled relative to neighboring cells within the population, the methodcomprising contacting the cell with a biomolecule of claim 17, whereinthe biomolecule comprises a targeting moiety that binds to a bindingpartner that is indicative of the cell, and thereby differentiallylabeling the cell relative to neighboring cells within the population.29. The method of claim 28, further comprises the step of imaging thecell, the imaging step comprising: i) directing exciting wavelength tothe cell; and ii) detecting emitted fluorescence from the cell.
 30. Themethod of claim 28, wherein the labeling takes place in vitro.
 31. Themethod of claim 28, wherein the labeling takes place in vivo.
 32. Amethod of labeling a polypeptide comprising: forming a complex thatcomprises the polypeptide and a binding agent, wherein the binding agentcomprises a fluorescent label derived from a compound according to claim1, wherein the at least one reactive moiety of Formula I has undergone areaction which attaches the label to the binding agent.
 33. The methodof claim 32, wherein the binding agent is an antibody.
 34. The method ofclaim 32, wherein the complex comprises (a) a primary antibody thatbinds to the polypeptide, and (b) the binding agent which functions as asecondary antibody exhibiting binding capability to the primaryantibody.
 35. The method of claim 32, wherein the labeling occurs on asolid substrate.
 36. The method of claim 32, wherein the labeling occursintracellularly.
 37. The method of claim 32, wherein the complex yieldsa signal to noise ratio greater than about
 100. 38. The method of claim32, wherein the signal to noise ratio is calculated by the formula:(fluorescent signal from a complex comprising the polypeptide bound by aprimary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).
 39. The method of claim32, wherein the complex yields a signal to noise ratio greater thanabout 250, wherein the signal to noise ratio is calculated by theformula:(fluorescent signal from a complex comprising the polypeptide bound by aprimary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).
 40. The method of claim32, wherein the complex yields a signal to noise ratio greater thanabout 270, wherein the signal to noise ratio is calculated by theformula:(fluorescent signal from a complex comprising the polypeptide bound by aprimary antibody which in turn is bound to the bindingagent)/(fluorescent signal from a mixture of the polypeptide, an isotypecontrol primary antibody and the binding agent).